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Immuno-gold labeling of EGF-receptors in cultured A431 cells
184
Abstracts ~STheNerherlands Sociery of
among muscle fibre types of the EDL and GM of the rat. Other studies using this technique do describe a difference in capillary/fibre ratio between slow oxiThe dative and fast glycolytic fibres. alkaline phosphatase seems to give unreliable results. The same muscle fibres have been studied using serial sections for alkaline phosphatase/ATP-ase histochemistry and electron microscopy. Cryosections, while still frozen, were immersed in 2oC glutaraldehyde fixative. The fixed sections were processed for EM. From ultrathin sections 4 fibres were randomly selected and all capillaries surrounding them were photographed. Compared with the histochemical stained sections, we found that of 19 capillaries (EM),17were stained with alkaline phosphatase, while at the (EM) location of fibrocytes, 3 in this case, histochemical staining was deOther LM staining procedures tected. for capillary detection, such as specific binding to Ulex europaeus, will be examined for their validity using ultramicroscopy.
Electron Microscopy
under it. Because of its localization, the gold label is not replicated. Following freeze-etching, replication and cleaning of the replicas, the gold particles are trapped in the replica. Essentially all gold particles show shadow cones, indicating that they are indeed replicated. Density of gold label in both label-fracture and freezeetch replicas varied between 75 and 300 particles/pm2. Characterization of the lateral distribution of gold label by means of a modified differential density distribution analysis revealed that the gold particles were clustered (p < 0.001). Upon treatment with 50 ug/ml EGF, clusterinq increased 1.5-fold within one minute, and then decreased to control levels within three minutes. Treatment of A431 cells with 50 pg/ml concanavalin A for 5 minutes resulted in clustering of both EGF-receptor and other transmembrane proteins (indicated by the clustering of intramembrane particles). IMMUNO-GOLD IN CULTURED
EFFECTS OF EPIDERMAL GROWTH FACTOR AND CONCANAVALIN A ON LATERAL DISTRIBUTION OF EGF RECEPTORS IN A431 CELLS, VISUALIZED BY LABEL-FRACTURE AND FREEZE-ETCH IMMUNOCYTOCHEMISTRY N. van Belzen, P.J. Rijken, W.J. Hage*, J. Boonstra and A.J. Verkleij Department of Molecular Cell Biology, University of Utrecht, Padualaan 8, and *Hubrecht Labor3584 CH Utrecht; 3584 CT Utrecht atory, uppsalalaan 8,
The lateral distribution of epidermal growth factor (EGF) receptors on A431 cells has been studied by two complementary methods: label fracture and freezeetching, both in combination with immunoA431 human epidermoid sold labelins. carcinoma ceils were consecutively labeledwithmonoclonal anti-EGF receptor antibodies, rabbit anti-mouse antibody and protein A-gold conjugate, frozen in solid/liquid nitrogen, freeze-fractured or freeze-etched, and replicated with a Pt/C according to standard procedures. Electron micrographs representing a replica area of 3.6 urn2 were digitized by means of an IBAS II interactive image analysis system (Kontron/Zeiss). After label-fracture, the replica of the exoplasmic fracture face can be observed in one single, coincident image with the gold label which is situated
LABELING OF EGF-RECEPTORS A431 CELLS
P. van Bergen en Henegouwen, J. Boonstra, N. van Belzen, P. van Maurik*, F.A.C. Wiegant and A.J. Verkley of Molecular Cell Biology, Dept. University of utrecht, Padualaan 8, *Hubrecht Labora3584 CH Utrecht; Uppsalalaan 8, 3584 CT Litrecht, tory, The Netherlands
The recent development of a number of methods (cryo-ultramicrotomy, freezeetching, label-fracture, surface replication and dry-cleavage) combined with immuno-gold labeling provides the possibility to study the distribution of cell-surface and intracellularly located antigens at the ultrastructural level. We have applied these methods in the visualization of EGF-receptors in A431 cells, using a monoclonal anti-EGFreceptor antibody designated 2E9, A dense labeling was observed on the plasma membrane and intracellularly on a network of tubular and vesicular membranes, multivesicular bodies, lysosomes and Golgi membranes. The method is therefore suitable for ultrastructural studies of EGF-receptor biosynthesis and receptor mediated endocytosis. An important feature of growth factor receptor interaction in the mitogenic response appears to be the redistribution of cell-surface-located receptors,
Abstracts
of i%
185
Netherlands Society ofElectron Microscopy
Freezeso-called receptor clustering. etching and label-fracture are both convenient methods to analyze the lateral distribution of cell-surface-located EGF receptors and therefore can be used to study clustering phenomena in detail. Finally, evidence has been obtained of an association of growth factor receptors and cytoskeletal elements. We have used surface-replication and drycleavage methods on immuno-labeled cells, in the presence or absence of Triton x-100, to visualize the association between EGF-receptors and cytoskeletal elements on an ultrastructural level.
models containing 1~10~~ M of the respective drugs served as controls. In contrast to findings in spleen and models, immunogold labels were rarely encountered in nerve sections, indicating minimal or absent penetration of the drugs in cells of healthy peripheral nerves. We acknowledge by
financial
support
of the work
NSL/ILEP grant nr. 8030015.
VARIATION IN THE DENSITY DISTRIBUTIONOF CRYOSECTIONS AND CONTRAST AT LOWTEMPERATURE OBSERVATION P.H.H. Bomans and P.M. Frederik
IMMUNO-GOLD TEM STUDIES ON THE INTRACELLULAR DEPOSITION OF ANTI-LEPROSY DRUGS (DDS, RIFAMPICIN) IN THE SPLEEN VERSUS PERIPHERAL NERVES J. Boddingius,
H. Dijkman
and M. De Wit*
Inst. Dermato-Venerology, Research UniLaboratory, Hoogbouw 20, Erasmus versi ty, Rotterdam, The Netherlands; *Royal Tropical Institute, Amsterdam, The Netherlands In multibacillary leprosy neuropathy, adequate penetration and accumulation of anti-leprosy drugs into Schwann cells and other intraneural host cells of Mycobacterium leprae could be impaired in certain nerve branches by intact "blood-nerve" and "perineurial" barriers and perhaps by the Schwann basal lamina. Low drug levels might give rise to drugresistance of some intraneural M.Leprae. Lack of drug penetration might lead to drug-sensitive viable bacilli surviving the currently applied short-term triplePublished data on intradrug treatment. cellular drug penetration in nerves are thus far lacking. By inununo-gold (TEM) methods, we investigated the intracellular presence of anti-leprosy drugs (DDS, Rifampicin) in the spleen and nerves of drug-fed healthy mice. Drug antibodies were raised in rabbits and employed as primary antibodies. Gold (10 nm)-labeled goat anti-rabbit IgG was utilized as secondary antibody. For DDS studies, etched ultrathin sections of glutaraldehyde- and osmiumtetroxidefixed and Araldite-embedded spleen and nerves were used besides ultracryosections of glutaraldehyde- or buffered For Rifampicin formalin-fixed tissues. studies, ultracryosections of tissues in a special EM fixative were employed. Immunolabeling of sections of gelatin
Dept. of Pathology, sity of Limburg, P. 6200 MD Maastricht,
EM-unit, Univer0. BOX 616, The Netherlands
Mildly fixed (glutaraldehyde 0.2%, formaldehyde 2%) tissues were infiltrated with glycerol (67% w/w) or propylene-glycol (80% w/w). These concentrations prevent formation of ice crystals and allow subcooling and vitrification at moderate cooling velocities. Moreover, even upon warming no crystallization will occur: the exit of the glass phase will lead to a sub-cooled liquid and thus only the viscosity will change. Tissues vitrified in these solutes are particularly suitable for cryo-sectioning and cryo-observation; a wide range of temperatures can be used to search the optimum sectioning conditions and the density difference between tissue components (e.g. protein 1.3) and embedding aqueous solutes (e.g. glycerol 1.17, propylene ylycol 1.04, ice 0.92) can be varied. In cryosections from glycerol-treated tissue no contrast was observed upon low temperature observation under low-density conditions. By replacing glycerol with propylene ylycol improved contrast was sought in increasing the difference between the mass density of protein (e.g. zymoyen yranules) and the surrounding aqueous environment. Although the scattering properties of propylene glycol approach those of (pure) water, no appreciable contrast was observed in low-dose uictures of cryosections from propyleneglycol infiltrated tissue. The ultrastructure only became evident after several exposures at low-dose conditions. There remains a possibility that in thick cryosections or by thickness variations in cryosections intrinsic contrast between subcellular organelles is
Abstracts ~STheNerherlands Sociery of
among muscle fibre types of the EDL and GM of the rat. Other studies using this technique do describe a difference in capillary/fibre ratio between slow oxiThe dative and fast glycolytic fibres. alkaline phosphatase seems to give unreliable results. The same muscle fibres have been studied using serial sections for alkaline phosphatase/ATP-ase histochemistry and electron microscopy. Cryosections, while still frozen, were immersed in 2oC glutaraldehyde fixative. The fixed sections were processed for EM. From ultrathin sections 4 fibres were randomly selected and all capillaries surrounding them were photographed. Compared with the histochemical stained sections, we found that of 19 capillaries (EM),17were stained with alkaline phosphatase, while at the (EM) location of fibrocytes, 3 in this case, histochemical staining was deOther LM staining procedures tected. for capillary detection, such as specific binding to Ulex europaeus, will be examined for their validity using ultramicroscopy.
Electron Microscopy
under it. Because of its localization, the gold label is not replicated. Following freeze-etching, replication and cleaning of the replicas, the gold particles are trapped in the replica. Essentially all gold particles show shadow cones, indicating that they are indeed replicated. Density of gold label in both label-fracture and freezeetch replicas varied between 75 and 300 particles/pm2. Characterization of the lateral distribution of gold label by means of a modified differential density distribution analysis revealed that the gold particles were clustered (p < 0.001). Upon treatment with 50 ug/ml EGF, clusterinq increased 1.5-fold within one minute, and then decreased to control levels within three minutes. Treatment of A431 cells with 50 pg/ml concanavalin A for 5 minutes resulted in clustering of both EGF-receptor and other transmembrane proteins (indicated by the clustering of intramembrane particles). IMMUNO-GOLD IN CULTURED
EFFECTS OF EPIDERMAL GROWTH FACTOR AND CONCANAVALIN A ON LATERAL DISTRIBUTION OF EGF RECEPTORS IN A431 CELLS, VISUALIZED BY LABEL-FRACTURE AND FREEZE-ETCH IMMUNOCYTOCHEMISTRY N. van Belzen, P.J. Rijken, W.J. Hage*, J. Boonstra and A.J. Verkleij Department of Molecular Cell Biology, University of Utrecht, Padualaan 8, and *Hubrecht Labor3584 CH Utrecht; 3584 CT Utrecht atory, uppsalalaan 8,
The lateral distribution of epidermal growth factor (EGF) receptors on A431 cells has been studied by two complementary methods: label fracture and freezeetching, both in combination with immunoA431 human epidermoid sold labelins. carcinoma ceils were consecutively labeledwithmonoclonal anti-EGF receptor antibodies, rabbit anti-mouse antibody and protein A-gold conjugate, frozen in solid/liquid nitrogen, freeze-fractured or freeze-etched, and replicated with a Pt/C according to standard procedures. Electron micrographs representing a replica area of 3.6 urn2 were digitized by means of an IBAS II interactive image analysis system (Kontron/Zeiss). After label-fracture, the replica of the exoplasmic fracture face can be observed in one single, coincident image with the gold label which is situated
LABELING OF EGF-RECEPTORS A431 CELLS
P. van Bergen en Henegouwen, J. Boonstra, N. van Belzen, P. van Maurik*, F.A.C. Wiegant and A.J. Verkley of Molecular Cell Biology, Dept. University of utrecht, Padualaan 8, *Hubrecht Labora3584 CH Utrecht; Uppsalalaan 8, 3584 CT Litrecht, tory, The Netherlands
The recent development of a number of methods (cryo-ultramicrotomy, freezeetching, label-fracture, surface replication and dry-cleavage) combined with immuno-gold labeling provides the possibility to study the distribution of cell-surface and intracellularly located antigens at the ultrastructural level. We have applied these methods in the visualization of EGF-receptors in A431 cells, using a monoclonal anti-EGFreceptor antibody designated 2E9, A dense labeling was observed on the plasma membrane and intracellularly on a network of tubular and vesicular membranes, multivesicular bodies, lysosomes and Golgi membranes. The method is therefore suitable for ultrastructural studies of EGF-receptor biosynthesis and receptor mediated endocytosis. An important feature of growth factor receptor interaction in the mitogenic response appears to be the redistribution of cell-surface-located receptors,
Abstracts
of i%
185
Netherlands Society ofElectron Microscopy
Freezeso-called receptor clustering. etching and label-fracture are both convenient methods to analyze the lateral distribution of cell-surface-located EGF receptors and therefore can be used to study clustering phenomena in detail. Finally, evidence has been obtained of an association of growth factor receptors and cytoskeletal elements. We have used surface-replication and drycleavage methods on immuno-labeled cells, in the presence or absence of Triton x-100, to visualize the association between EGF-receptors and cytoskeletal elements on an ultrastructural level.
models containing 1~10~~ M of the respective drugs served as controls. In contrast to findings in spleen and models, immunogold labels were rarely encountered in nerve sections, indicating minimal or absent penetration of the drugs in cells of healthy peripheral nerves. We acknowledge by
financial
support
of the work
NSL/ILEP grant nr. 8030015.
VARIATION IN THE DENSITY DISTRIBUTIONOF CRYOSECTIONS AND CONTRAST AT LOWTEMPERATURE OBSERVATION P.H.H. Bomans and P.M. Frederik
IMMUNO-GOLD TEM STUDIES ON THE INTRACELLULAR DEPOSITION OF ANTI-LEPROSY DRUGS (DDS, RIFAMPICIN) IN THE SPLEEN VERSUS PERIPHERAL NERVES J. Boddingius,
H. Dijkman
and M. De Wit*
Inst. Dermato-Venerology, Research UniLaboratory, Hoogbouw 20, Erasmus versi ty, Rotterdam, The Netherlands; *Royal Tropical Institute, Amsterdam, The Netherlands In multibacillary leprosy neuropathy, adequate penetration and accumulation of anti-leprosy drugs into Schwann cells and other intraneural host cells of Mycobacterium leprae could be impaired in certain nerve branches by intact "blood-nerve" and "perineurial" barriers and perhaps by the Schwann basal lamina. Low drug levels might give rise to drugresistance of some intraneural M.Leprae. Lack of drug penetration might lead to drug-sensitive viable bacilli surviving the currently applied short-term triplePublished data on intradrug treatment. cellular drug penetration in nerves are thus far lacking. By inununo-gold (TEM) methods, we investigated the intracellular presence of anti-leprosy drugs (DDS, Rifampicin) in the spleen and nerves of drug-fed healthy mice. Drug antibodies were raised in rabbits and employed as primary antibodies. Gold (10 nm)-labeled goat anti-rabbit IgG was utilized as secondary antibody. For DDS studies, etched ultrathin sections of glutaraldehyde- and osmiumtetroxidefixed and Araldite-embedded spleen and nerves were used besides ultracryosections of glutaraldehyde- or buffered For Rifampicin formalin-fixed tissues. studies, ultracryosections of tissues in a special EM fixative were employed. Immunolabeling of sections of gelatin
Dept. of Pathology, sity of Limburg, P. 6200 MD Maastricht,
EM-unit, Univer0. BOX 616, The Netherlands
Mildly fixed (glutaraldehyde 0.2%, formaldehyde 2%) tissues were infiltrated with glycerol (67% w/w) or propylene-glycol (80% w/w). These concentrations prevent formation of ice crystals and allow subcooling and vitrification at moderate cooling velocities. Moreover, even upon warming no crystallization will occur: the exit of the glass phase will lead to a sub-cooled liquid and thus only the viscosity will change. Tissues vitrified in these solutes are particularly suitable for cryo-sectioning and cryo-observation; a wide range of temperatures can be used to search the optimum sectioning conditions and the density difference between tissue components (e.g. protein 1.3) and embedding aqueous solutes (e.g. glycerol 1.17, propylene ylycol 1.04, ice 0.92) can be varied. In cryosections from glycerol-treated tissue no contrast was observed upon low temperature observation under low-density conditions. By replacing glycerol with propylene ylycol improved contrast was sought in increasing the difference between the mass density of protein (e.g. zymoyen yranules) and the surrounding aqueous environment. Although the scattering properties of propylene glycol approach those of (pure) water, no appreciable contrast was observed in low-dose uictures of cryosections from propyleneglycol infiltrated tissue. The ultrastructure only became evident after several exposures at low-dose conditions. There remains a possibility that in thick cryosections or by thickness variations in cryosections intrinsic contrast between subcellular organelles is