Biological photoemission microscopy at maximum

216

Cell Biology

USING ARTIFICIAL CONSTRUCTIONS MODELS OF VIRUSES FOR SELECTIVE TARGETING OF GENES INTO LIVING CELLS Alexander S.Sobolev, Andrei A.Rosenkrsnz, Sergei V.Yachmenev, Research Center of Molecular Diagnostics, USSR Ministry of Health, Moscow, USSR Artificial constructions have been developed, possessing three properties of virian ability to reversibly bind ons, namely, nucleic acids, to recognize internalizable receptors on the cell surface and translocate into the nucleus. By an example of one of such constructions containing DNA (a plasmid PSVRP-8 encoding a large T-antigen SV-40, missing in human cells) in the complex with a polycation (poly-L-lysine) covalently linked with the membrane receptor ligand (insulin) it was demonstrated that such models of viruses are capable to penetranslocate into the nuctrate into cells, cleus and transport genetic material into the latter. The efficacy of transfection comprised 0.95t 0.16 % for human hepatoma The addition of free PLC/PRF/5 cell line. insulin to cells reliably inhibited transwhich is indicative of the involvefection, ment of hepatoma cell insulin receptors in the recognition of the construction and its transport via receptor-mediated endocytosis

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A PREPARATION METHOD DF CELL DYNAMICS: Gerd Knoll and Helmut Plattner, Biologie, Universitgt Konstanz, D-7750 Konstanz, FRG

FOR THE QUENCHED Fakultgt Postfach

ANALYSIS FLW fiir 5560,

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lipids

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Christina Schlatterer ftir Biologie, Universitst D-7750

Konstanz,

TIK

Supplement

OF CA-INDICATOR f4EASURE!4ZNTS

Dieter

and

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Malchow, Postfach

Konstanz,

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the cytoplasm to these cells. After the

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that

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Upon chemotactic stimulation of Dictyostelium discoideum amebae extracellular calcium is by the cells (Bumann et al., 1984, J. Cell 98:173-178). In order to measure intracellular calcium we introduced the free acid of the

indicator

1990

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an

of

the

up

calcium aequorin into

photoprotein

injection

taken Biol.

technique

adapted

we were confronted with the the indicators were rapidly

sequestered and extruded from the cytoplasm. Sequestration of injected molecules seems to be a common phenomenon of different cell types. The kinetics and specificities of this process are not clear. Nevertheless there are molecules as dextran and its derivatives, that are not sequestered (Luby-Phelps et al., 1986, J. Cell Biol. 102:2015-2022). Therefore we covalently linked furaand indo-l to dextran. In fact this modification effectively prevents the sequestration of the dyes from the cytoplasm. In the physiological calcium range the calcium-binding and fluorescence characteristics of the coupled dyes approach the original molecules to 90 X. With these modified indicator molecules long time measurements of calcium in intact cells are possible. Supported by SFB 156.

BIOLOGICAL De Stasio.

lstituto

PHOTOEMJSSION AT MAXIMUM di Strutturn

MICROSCOPY

dslla Materia del CNR, 00044

Roma, Italy.

Electron parallel have been

Vol. 14, Abstracts

A MODIFICATION FOR LONG

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Gelsomina

cells.

Cells once frozen have been variety of analytical methods. allows for thin section, freeze label and element analysis. In

Reports,

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Rapid responses of living cells demand for adequate time resolution of analytical methods. This problem is most obvious for “non-real time” approaches like electron microscopy and biochemistry. On the basis of established methods in (bio)chemistry (Chance, El. et al., 1964, Rapid Mixing and Sampling Techniques in Biochemistry. Academic Press, New York and London) we therefore developed a preparation procedure for subsequent analysis of intact cells in defined states with high time resolution. Cells are synchronously stimulated by a suitable compound during continuous flow through a mixing chamber. After a defined time period the suspension

is frozen in liquid propane. The system is optimized for 1. high time resolution (in the of milliseconds) and 2. for full integrity of

International

a

by biochemical means. (his combination of techniques has been applied to define and to correlate dynayic aspects of exo-endocytosis coupling in Paramecium cells. These processes normally cannot be distinguished from each other, since coupling requires only= 300 ms. Supported by SFB 156.

W. Ng, A. K. Ray-Chaudhuri, R. K. Cole, 2. Y. Guo, J. Wallace, G. Margaritondo and F. Cerrina. University of Wisconsin, Madison, Wisconsin 53706. J. Underwood, R. Perera and J. Korlright. Center for X-Ray Optics, Lawrenoa Berkeley Laboratory, Berkeley, Caliiomia 94720. Delio Meroanti and M. Teresa Ciittl. lstltuto di Neurobiologia del CNR, 00100 Roma, Italy. We recently obtained the first scanning photoemission micrographs of biological samples, using MAXIMUM, the undulator photoemission spectromicrosoope at the Wisconsin Synchroiron Radiation Center; this opens the way to novel investigation techniques in the life sciences, such as the microanalysis of the chemical status of component elements. Uncoated and u&belled neurons were imaged after having been prepared with a new technique suitable for ultrahigh vacuum experiments. Photoemission spectromicroscopy is an emerging technique, that is made possible by high-brightness undulator sources of synchrotron radiation. and will be enhanced in the future by the advent of ultrahigh-brightness sources such as ELElTRA atTrieste. As it is known, conventional photoemission spectroscopy (e.g., ESCA) delivers infonatlon on the chemical elements of the specimen, and on their chemical status. Photoemission spectromicrosoopy has the unique capability of delllring the same kind of information for microscopic areas. For example, it can provide micrographs of a specific oxidation state of a specific element. It is, therefore, complementary to other mlcroscoples and spe&oscopies. The successful tests here described demonstrate that ca#ular soecimens ohotoemit orovidino sufficient contrast, and also that the’prepara&h technique does tiot alter the external structure of the neuronsMAXIMUM has the capability of providing visible micrographs besides the photoemission micrographs; this was used to test the shape corfespondance for the same region of the specimen, and also the differences atiaing from the surface sensitivity of the photoemission probe. Possible applications will be discussed. with particular emphasis on trace element problems such as Ca channel quantification in cell membranes, and local chemical analysis related to neural pulse transmission.