22nd Meeting of DSFCM

 

Applications of Green Fluorescent Protein in Biology and Medicine

 

Joint symposium of the Danish Society for Flow Cytometry and the Danish Society for Biochemistry and Molecular Biology

 

27 April 2000, 12:00-17:00

Auditorium 2, Rigshospitalet, Blegdamsvej 9, Copenhagen

 

Organizers/chairmen:

Carl-Henrik Brogren and Jørgen K. Larsen (DSFCM, www.flowcytometri.dk), and Steen Gammeltoft DSBMB, www.biokemi.org)

 

Sponsors:

The Danish Medical Society, BD (Becton Dickinson/Clontech), and RAMCON A/S (Beckman Coulter)

 

All are welcome!

 

Program:

 

 12:00-12:50

Janet Jansson, Section for Natural Sciences, Södertörns Högskola, Huddinge, Sweden

Use of GFP to monitor specific bacterial populations in environmental samples

 

12:50-13:40

Raphael H. Valdivia, University of California, Berkeley

Bacterial Genetics and flow cytometry: Novel approaches to the study of bacterial pathogenesis

 

13:40-14:10     

Ole Thastrup, BioImage A/S

The usage of green fluorescent proteins in drug discovery

 

14:10-14:30     

Pause - Sandwiches/refreshments

 

14:30-14:50     

Lene Martini and Kristian Kirk Jensen, Laboratory of Molecular Pharmacology, Panum Institute, University of Copenhagen

Green fluorescent protein in studies of 7 transmembrane receptors

 

14:50-15:10     

Bjarke Bak Christensen, Claus Sternberg, Jens Bo Andersen, Janus Haagensen, and Søren Molin, Department of Microbiology, Technical University of Denmark

Applications of GFP as an in situ marker of plasmid transfer, and microbial activity in biofilms

 

15:10-15:30     

Bo Normander, Department of Marine Ecology and Microbiology, National Environmental Research Institute

GFP as a reporter of bacterial distribution, activity and gene transfer in the plant environment

 

15:30-15:50     

Pause - Coffee/tea/cakes

 

15:50-16:05     

Solveig Krogh Christiansen, Department of Plant Biology and Biogeochemistry, Risø National Laboratory

GFP expression in an obligate plant pathogenic fungus

 

16:05-16:20     

Uffe Birk Jensen and Lars Bolund, Institute of Human Genetics, University of Aarhus

Use of GFP as a reporter gene in the study of the efficacy of non-viral gene transfer to epidermal cells

 

16:20-16:35     

Tine U. Sørensen, Laboratory for Infectious Diseases 144, Hvidovre Hospital

Simultaneous use of three different fluorescent proteins as transduction markers in gene therapy research

 

16:35-16:50     

Carl-Henrik Brogren and Susana Alvarez Herrero, Institute of Food Safety and Toxicology, Danish Veterinary and Food Administration

Salmonella-GFP in macrophage phagocytosis

 

16:50-17:00     

Pause - Refreshments

 

17:00-

General assembly of the Danish Society for Flow cytometry

 

 

ABSTRACTS

 

Use of GFP to monitor specific bacterial populations in environmental samples

Janet K. Jansson. Section for Natural Sciences, Södertörns högskola, Box 4101, 141 04 Huddinge, Sweden. Tel.: +46-8-585 88744. Fax: +45-8-585 88510. Email: janet.jansson@sh.se

The use of bacterial inoculants for different environmental applications is becoming increasingly popular. For example, bacteria can be used to prevent plant diseases (biocontrol) or to degrade toxic pollutants (bioremediation). We are interested in monitoring of bacterial inoculants in environmental samples in order to determine their numbers, activity, distribution and mode of action. Bacteria of interest were tagged with marker genes so that they could be specifically identified amidst members of the natural mixed microbial community. Some strains were chromosomally tagged with one or two copies of the gfp gene, encoding GFP (green fluorescent protein). The gfp-tagged strains were monitored by flow cytometry and by different fluorescence microscopy techniques in soil and/or on plant surfaces. In some cases, the cells were additionally tagged with the luc or luxAB genes encoding luciferase enzymes. Luciferase activity was used as an indicator of cell metabolic activity, whereas GFP fluorescence was used for enumeration of the total number of cells, regardless of their activity. Flow cytometry was found to be an excellent tool for enumeration of gfp-tagged cells in soil and in plant homogenates, however the background level of fluorescent particles was considerable. We incorporated the use of Nycodenz density gradient centrifugation as a method to separate the bacterial cell fraction from soil particles or plant homogenates before injection into the flow cytometer. By incorporation of an internal standard of microscopic beads, the number of fluorescent cells in the samples were accurately enumerated. Usually, the number of cells counted by flow cytometry was greater than that enumerated by selective plate counting. Therefore, a proportion of the cells counted by flow cytometry are non-culturable or possibly even dead cells. We are currently investigating the reason for this discrepancy.

 

Bacterial genetics and flow cytometry: Novel approaches to the study of bacterial pathogenesis

Raphael H. Valdivia. Department of Molecular and Cell Biology, 634 Barker Hall, University of California, Berkeley, CA 94720, U.S.A. Tel.:+1 510-642-5756. E-mail: valdivia@uclink4.berkeley.edu

Salmonella typhimurium survives and replicates in the intracellular environment of a variety of mammalian cells by activating the transcription of bacterial factors that block the destruction of the organism by lysosomal enzymes, protect the bacterium from antimicrobial peptides, and provide the nutrient-scavenging capabilities to survive in an intracellular vacuole. In order to further our understanding of the molecular and genetic basis of the interaction between S. typhimurium and its host cell, we have developed a flow sorting-based gene selection strategy to identify S. typhimurium bearing gfp gene fusions that are preferentially expressed in the intracellular environment of host cells. In this manner, bacteria were separated by fluorescence-activated cell sorting on the basis of stimulus-dependent fluorescence induction. This selection technology, termed differential fluorescence induction (DFI) permitted the isolation of eight genes expressed in response to low pH and fourteen genes preferentially expressed in the macrophage environment. These macrophage-inducible genes (mig) were found to code for cell envelope proteins, cell-surface maintenance enzymes, stress response proteins, transcriptional activators, and a component of a type III secretion system required for virulence. All mig analyzed were found to be expressed in the intracellular environment of a variety of non-macrophage cells, and in the splenocytes and hepatocytes of infected animals. DFI selections were also used to identify regulatory loci for mig. Intracellular expression of migs was found to be dependent on at least four different genetic regulatory systems including PhoP/PhoQ, OmpR/EnvZ, PmrA/PmrB and SsrA/SsrB. Other applications of flow sorting in the study of host-pathogen interaction will be discussed.

 

The usage of Green Fluorescent Proteins in Drug Discovery

Ole Thastrup. BioImage A/S, Moerkhoej Bygade 28, DK-2860 Soeborg, Denmark. Tel.:+45 4443-7517. Fax:+45 4443-7505. E-mail: ot@bioimage.dk. URL: www.bioimage.dk

Green Fluorescent Proteins (GFPs) have opened for a wealth of new opportunities in drug discovery. GFPs thus allow development of new assay systems that in physiologically correct scenarios are capable of screening compound libraries against previously intractable drug targets.

The pharmaceutical industry is under a considerable pressure to utilize the constantly growing resource of potential drug targets coming out of genomics. Essential to a successful exploitation is the creation of novel ways to analyse for the functional characteristics of these targets and a fast development of robust drug screening systems.

In BioImage A/S, a recent spin-out of Novo Nordisk, we have developed a broad proprietary discovery platform, which allows us to screen against various families of drug targets involved in intracellular signalling. Important components of our screening systems are GFPs engineered to monitor signalling events that are involved in the redistribution of specific signalling components. GFPs have proven indispensable in the characterisation of these important signalling phenomena and in the identification of compounds that are capable of modulating them.

GFPs from various marine organisms, and derivatives thereof, have already revolutionised the way functional cell biology is conducted, it is most likely that these genetic tools also will form the basis for a paradigm shift in the drug discovery industry.

 

Green Fluorescent Protein Studies of 7 Transmembrane Receptors

Lene Martini and Kristian Kirk Jensen

Lab. for Molecular Pharmacology, Dept. Pharmacology, The Panum Institute, Build 18.6,

University of Copenhagen, DK-2200 Copenhagen

Email: martini@molpharm.dk and kirk@molpharm.dk

The superfamily of 7 transmembrane receptors comprises the largest group of membrane proteins with numerous important physiological functions, and is therefore a key target in drug development.

Signalling through the receptor commences by the binding of a ligand to its extracellular domains. Following conformational changes it activates specific members of the signal transducing guanine nucleotide-binding regulatory proteins, the G proteins, on the cytoplasmic side of the cell membrane. In turn these modulate the activity of down-stream-effectors, thereby changing the physiology of the cell. In order to turn off this signalling-cascade, a cytosolic protein termed arrestin is recruited to the membrane, where it has a dual role both terminating the G protein signalling and mediating receptor internalisation, thereby removing it from any external stimuli.

For many years the molecular structure and function of 7 transmembrane receptors has been elucidated mainly through mutagenesis studies. Their interaction with accessory proteins has in addition to mutagenesis been detected through the use of appropriately derived antibodies. Now the revolutionary discovery of GFP has made it possible to study the kinetics of the receptor and its associated proteins in real time in living cells by fluorescence microscopy. In addition, the synthesis of spectral variants of GFP, such as the cyan (CFP) and yellow (YFP) mutants, has made it possible to study the interactions and the kinetics of multiple labelled proteins simultaneously.

In our study, numerous chimeric proteins were synthesised comprising the neurokinin receptor, NK1, fused to CFP at various sites, and the following radioligand binding and functional assays assessed their performance. Also chimeric proteins consisting of beta-arrestin fused to YFP, were made. Combinations of one type of labelled receptor and one type of labelled beta-arrestin were then transiently co-expressed in COS7 cells and observed by epifluorescence microscopy prior to and after treatment with the ligand, Substance P. Thereby, the relative spatial and temporal distribution of the two proteins may be taken as another measure of receptor performance.

 

Applications of GFP as an in situ marker of plasmid transfer, and microbial activity in biofilms

Bjarke Bak Christensen, Claus Sternberg, Jens Bo Andersen, Janus Haagensen, and Søren Molin. Department of Microbiology, Technical University of Denmark, Building 301, DK-2800 Lyngby, Denmark. E-mail: bbc@fdir.dk

During the last 5-10 years the development of in situ markers for analysis of microbial communities, has evolved tremendously. In particular, the use of fluorescent in situ rRNA hybridization (FISH) for identification and localization of specific species combined with Gfp as an in situ reporter for gene expression has proven to be extremely successful. At the Microbial ecology group (DTU) we have used this combination of biomarkers in studies of microbial interactions in biofilms.

Different types of microbial interaction have been studied like: i) metabolic interactions, ii) communication mediated by Acyl-Homoserine-Lactone (AHL) signal molecules, and iii) plasmid transfer. For example, in a Benzyl alcohol degrading biofilm, combining quantitative determination of donor, recipient and transconjugants with in situ monitoring of single cells through zygotic interaction of Gfp fluorescence provided hitherto unknown details about spread of the TOL-plasmid in the biofilm. In the same biofilm, we also studied metabolic interactions between the two dominant species (P. putida RI and Acinetobacter C6). By inserting, into P. putida RI, a cassette carrying the growth phase regulated ribosomal RNA promoter rrnB P1 fused to an unstable variant of the gfp gene it was possible to monitor the metabolic activity of P. putida RI at different locations in the biofilm. The activity of P. putida RI was shown to be higher near micro-colonies of Acinetobacter C6, which was further shown to be caused by leakage of the metabolic intermediate, benzoate, from Acinetobacter C6 into the surrounding where it is degraded by P. putida RI. This metabolic cross talk turned out to have a significant impact on the structural organization of the two species in the biofilm.

Recently a number of new monitor strains for in situ detection of various AHL signal molecules has been developed. These are based on a cassette carrying the luxR gene and the luxI promoter (or homologues to lux) fused to gfp. Thus, in the presence of high concentrations of AHL the monitor strain becomes green fluorescent. Preliminary studies have shown that the production of signal molecules in large micro-colonies in biofilms may have a significant impact on development of other micro-colonies in the regions near the large micro-colonies.

 

GFP as a reporter of bacterial distribution, activity and gene transfer in the plant environment

Bo Normander. Department of Marine Ecology and Microbiology, National Environmental Research Institute, DK-4000 Roskilde, Denmark. Tel.:+45 4630-1244. Fax:+45 4630-1216. E-mail: bn@dmu.dk

The green fluorescent protein (GFP) has proved to be a powerful tool in in situ studies of bacteria introduced into the terrestrial environment. By confocal laser scanning microscopy (CLSM), the spatial localisation of the gfp-tagged bio-control strain, Pseudomonas fluorescens DR54-BN14 in the barley rhizosphere was studied. Within short distances (<30 µm) on the root surface, DR54-BN14 varied in size from small coccoid cells to large dividing rod-shaped cells. Also, it was found that DR54-BN14 was closely associated with the indigenous bacteria and commonly situated near or in the crevices between the epithelial root cells. A micro-colony assay, involving the enumeration of single cells and cells forming micro-colonies, showed a high viability of DR54-BN14 on roots. However, the activity of DR54-BN14 on roots, as measured by image-analysis of single cells, was low and comparable to the activity of starved cells. Finally, by using GFP it has been possible to detect hot-spots for conjugal gene transfer on plant leaves.

 

GFP expression in an obligate plant pathogenic fungus

Solveig Krogh Christiansen. Department of Plant Biology and Biogeochemistry, Risø National Laboratory, DK-4000 Roskilde, Denmark. E-mail: solveig.christiansen@risoe.dk

The obligate parasitic fungus, Blumeria graminis f.sp. hordei (Bgh), causing the barley powdery mildew disease reduces grain yield in all temperate climate zones. Huge amounts of fungicides are used to control the disease. The inhibition of specific processes rather than whole organisms will have less impact on the environment and our research aims at providing alternative strategies for management of the disease. GFP reporter gene technology provides a unique tool for studying the infection process of obligate parasites because it is based on a non-destructive assay. Using differential screening techniques we have identified a number of stage specific Bgh genes that are expressed during the infection process. To analyse the function of novel genes it is essential to know exactly in which cells and when in the infection process the genes are expressed. This can be accomplished in transgenic Bgh where a translational fusion has been made between the gene of interest and the GFP gene. Conidia from transgenic Bgh colonies will be transferred to new leaves and the cellular expression profile will be recorded following the infection process with a fluorescence microscope coupled to a camera. Among the most interesting are genes expressed in the fungal feeding organ, the haustorium, inside the barley epidermal cells. The in vivo expression pattern of these genes can only be determined using a Confocal Lazer Scanning Microscope in combination with the GFP reporter gene. Fungal gene products that are necessary for infection permit specific control measures to be developed in the form of targeted pesticides.

 

Transfection mediated cell cycle arrest in human epidermal keratinocytes

Uffe Birk Jensen and Lars Bolund. 

Institute of Human Genetics, The Bartholin Building, University of Aarhus, DK-8000 Aarhus C, Denmark

We have previously shown that one problem with non-viral gene transfer into human primary epidermal keratinocytes is cell cycle arrest of the productively transfected cells.

This was independent on vector construct and the p53 levels were unaffected by the procedures. Independence of p53 function was verified by using the keratinocyte cell line HaCat, mutated in both p53 alleles, which showed a marked reduction in clonogenic potency upon transfection. There was a slight increase of TUNEL positive apoptotic nuclei in the positive population at early time points. However, the apoptotic index was still very low. When we measured the frequency of involucrin expressing cells we found an increase in the productively transfected population over time indicating an initiation of terminal differentiation. In contrast to the transfected cultures, keratinocytes that were transduced using a retroviral vector showed no decrease in colony forming efficiency.

We are currently investigating the role of INK4 family and CIP/Kip family members in the observed phenomenon and using DNA labelled with dyes before transfection we are analysing the role of DNA entry into the cells as the precipitating factor.

 

Simultaneous use of three different fluorescent proteins as transduction markers in gene therapy research.

Tine U. Sørensen, Laboratory for Infectious Diseases 144, Hvidovre Hospital, Kettegård Allé 30, DK-2650 Hvidovre.

Abstract

By incorporating the gene encoding GFP in the vector used for transduction successfully transduced cells can be identified and sorted on the flow cytometer due to their green fluorescence. For many purposes different markers that can be used simultaneously are required. We have outlined a project requiring three different markers and are trying to find a combination of three different fluorescent proteins for use at the FACS Vantage flowcytometer. GFP and the yellow fluoreescent protein YFP are both effectively excited by the 488 nm laser, and even though their emission spectra are slightly overlapping it is possible to distinguish between the two by using appropriate filters and electronic compensation. So far we have not found a suitable third marker, but our candidates are the blue fluorescent protein, BFP and the newly available red fluorescent protein, DsRed. BFP is excited by the UV-laser and emits at a wavelength easily distinguished from the green and yellow fluorescence. DsRed has excitation optimum at 558 nm, but the excitation by the 488 nm laser should be sufficient for detection on the FACS. The emission of red light can easily be distinguished from the green and yellow light.

 

Salmonella-GFP in macrophage phagocytosis

Carl-Henrik Brogren and Susana Alvarez Herrero. Division of Microbiological Safety and Toxicology. Institute of Food Safety and Toxicology, DK-2860 Søborg, Denmark. E-mail: chb@fdir.dk

The GFP-gene inserted in the plasmids or chromosome of the bacteria has been used as reporter-gene system to study many problems related to clinical, food and environmental microbiology. However, various aspect of immunity against micro-organisms can also gain from this technology. The macrophage uptake of bacteria can be easily visualized and quantitated using GFP-labelled bacteria. This presentation concerns our attempt to measure the functional activity of chicken macrophages exposed to Salmonella-GFP. Two different video-imaging systems were applied for epifluorescense microscopy visualization of phagocytosis, an electronically intensified CCD system (Hamamatsu 2400-97), which allowed real-time recording, and a time-integrating 3-CCD colour system (SONY), which allowed low fluorescence still-picture recording. Both systems allowed manual recording of number and speed for Salmonella-GFP uptake in macrophages, but were both time-consuming methods. Consequently, we adapted our Beckman-Coulter Epics-XL/MCL flow cytometer to measure the Salmonella-GFP uptake in an automated and fast manner. This approach allowed a quantification of both active and non-active macrophages in phagocytosis, but we could not specifically subdivide the active population according to number of Salmonella-GFP bacteria phagocytized per macrophage. However, this was possible with fluorescent micro spheres. Differences in GFP-expression in the individual bacterial cells and degradation of the expressed GFP protein to different extent by intracellular proteolysis enzymes caused a broader range of fluorescence from macrophages with phagocytized Salmonella-GFP. However, the flow cytometric assay could give a semiquantitative estimate of phagocytic capacity and be used for time-course studies as well. The importance of these assays for elucidation of the phagocytosis and study effects of the related preventive immune reactions against Salmonellosis is obvious.