Genetically targeted staining of cells with voltage sensitive dyes using an ecto-enzyme [Elektronische Ressource] / Marlon Jakob Hinner

Frank Overton

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2005
• Nombre de lectures: 18
• Langue: English
• Poids de l'ouvrage: 18 Mo

Extrait

Max-Planck-Institut für Biochemie
Abteilung Membran- und Neurophysik



Genetically Targeted Staining of Cells with Voltage
Sensitive Dyes using an Ecto-Enzyme


Marlon Jakob Hinner





Vollständiger Abdruck der von der Fakultät für Chemie
der Technischen Universität München zur Erlangung des akademischen Grades eines


Doktors der Naturwissenschaften


genehmigten Dissertation.







Vorsitzender: Univ.-Prof. Dr. O. Nuyken
Prüfer der Dissertation: 1. Hon.-Prof. Dr. P. Fromherz
2. Univ.-Prof. Dr. T. Bach
3. Univ.-Prof. Dr. M.-E. Michel-Beyerle, i.R.



Die Dissertation wurde am 22.12.2004 bei der Technischen Universität München eingereicht
und durch die Fakultät für Chemie am 8.03.2005 angenommen.
Abstract

Fast Voltage Sensitive Fluorescent Dyes are membrane-bound, optical probes of membrane
potential. They are used to measure voltage transients in nerve cells. Recording signals from
individual cells in tissue requires selective staining of these cells. In this work, a novel
approach to this unsolved issue is presented. It relies on non-binding dye precursors that are
locally activated to bind to cell membranes by the hydrolytic action of a selectively
overexpressed, membrane bound enzyme.

Based on the structure of the common voltage sensitive hemicyanine dye Di-4-ASPBS, a
number of dyes with additional alcohol residues were synthesized. These were introduced
either at the hydrophilic headgroup appendix or at the lipophilic tail of the amphiphilic dyes.
By further reaction of the alcohol moieties to phosphate groups, potential dye precursors for
enzyme induced binding were obtained.
It was shown that phosphorylation of the headgroup appendix reduced membrane binding
by a factor of 16 to 22 for various dyes. Phosphorylation at the lipophilic tail reduced binding
drastically by a factor of 1000 to 10000.
An enzymatic assay revealed that all phosphate containing dyes were quantitatively
hydrolysed to the respective alcohols by Alkaline Phosphatase from the Human Placenta
(PLAP). Using this reaction, fluorescent dye binding activation to model membranes was
studied with soluble PLAP and small lipid vesicles, giant lipid vesicles or red blood cells.
To obtain a membrane-bound and plasma-membrane targeted construct of PLAP, the gene
of a fusion protein of soluble PLAP and an artificial membrane anchor was cloned. This
construct was overexpressed in the adherent mammalian cell lines HEK293 and MDCK, and
its correct targeting and functionality was ascertained by immunocytochemical and
histochemical methods.
Incubation of phosphatase expressing cells with dye precursors led to staining of their cell
membrane by enzymatic activation of dye binding. Selective staining of phosphatase-
expressing cells was successfully implemented when transfected and non-transfected cells
were cultured together and incubated with precursor dye.
In accordance with a theoretical model of the reaction, the prerequisites of selective staining
were a very strong membrane binding of the produced dye and a sufficiently large difference
in binding strength compared to the precursor dye.

Contents

1 Introduction...................................................................................1
1.1 Current Neurobiology ................................................................................1
1.2 Voltage Sensitive Dyes – A Tool in Need of Improvement .............................1
1.3 Enzyme Induced Selective Staining.............................................................3
1.4 Thesis Overview........................................................................................4
2 Modified Voltage Sensitive Dyes and their Membrane Interaction .......7
2.1 Synthesis and Spectroscopic Properties.......................................................7
2.2 Lipid Binding........................................................................................... 13
2.3 Electrostatic Influence on Binding 22
2.4 Membrane Permeation............................................................................. 25
2.5 Orientation of Tail-Modified Dye in Membranes.......................................... 28
2.6 Materials and Methods 30
3 Enzyme Induced Staining of Membranes by a Soluble Enzyme .........37
3.1 Enzymatic Hydrolysis of Phosphorylated Dyes............................................ 37
3.1.1 Soluble PLAP Accepts all Dye Substrates .......................................... 37
3.1.2 Hydrolysis Kinetics of Di-4-ASPPP measured by ITC .......................... 39
3.2 Enzyme Induced Staining of Liposomes..................................................... 40
3.3 nning of Giant Vesicles................................................ 44
3.4 Enzyme Induced Staining of Erythrocyte Membrane................................... 46
3.5 Materials and Methods............................................................................. 48
4 Genetic Targeting of an Enzyme to the Plasma Membrane...............51
4.1 Background ............................................................................................ 51
4.2 Plasma Membrane Targeting Signal for the Construction of Chimeras .......... 53
4.2.1 Design of the Construct ArtPlasMA................................................... 53
4.2.2 Gene Synthesis of ArtPlasMA........................................................... 55
4.3 Chimera of ArtPlasMA and PLAP (ArtPlasMA AP)......................................... 57
4.3.1 Immunocytochemical Detection – Western Blotting........................... 58
4.3.2 ochemical Detection – Immunofluorescence..................... 59
4.3.3 Histochemical Detection.................................................................. 62
4.3.4 Activity Determination of HEK293 Stably Expressing ArtPlasMA AP ..... 62
4.4 Conclusion.............................................................................................. 64
4.5 Materials and Methods............................................................................. 66

i 5 Cell Activated Staining with a Voltage Sensitive Dye........................71
5.1 Enzyme Induced Staining on Stably Phosphatase Expressing HEK293.......... 71
5.1.1 Di-10P-ASPBS ................................................................................ 71
5.1.2 Di-12P-ASPBS 73
5.2 Enzyme Induced Staining on Stably Expressing HEK293 vs. Native Cells ...... 74
5.2.1 Di-10P-ASPBS 74
5.2.2 Di-12P-ASPBS 74
5.2.3 Staining and Destaining after Removal of the Dye Precursor.............. 76
5.3 Enzyme Induced Staining: Profiles of Dye Diffusion.................................... 77
5.3.1 Di-10P-ASPBS 77
5.3.2 Di-12P-ASPBS 77
5.4 Enzyme Induced Staining on MDCK Transiently Expressing Phosphatase...... 80
5.5 Model of Enzyme Induced Staining ........................................................... 82
5.6 Discussion .............................................................................................. 84
5.7 Materials and Methods............................................................................. 89
6 Final Conclusion and Outlook.........................................................91
7 Appendix .....................................................................................95
7.1 Additional Experiments ............................................................................ 95
7.1.1 A Study on Gene Synthesis: LCR vs. PCR and the Origin of Mutation .. 95
7.1.2 Comparison of an Artificial and a Natural Targeting Signal............... 110
7.2 Expanded Background ........................................................................... 117
7.2.1 Lipids and Vesicles ....................................................................... 117
7.2.2 Human Alkaline Phosphatases....................................................... 119
7.3 Tables.................................................................................................. 121
7.3.1 Human and Yeast Codon Usage .................................................... 121
7.3.2 Oligonucleotides used for the Synthesis of ArtPlasMA...................... 121
7.3.3 Olig for theAsglypMA 122
7.3.4 Oligonucle for theArtPlasMA sine TM.......... 122
7.4 Vector Maps ......................................................................................... 123
7.5 Abbreviations........................................................................................ 124
7.5.1 General Abbreviations................................................................... 124
7.5.2 Nomenclature of the Dyes ............................................................ 125
8 Literature................................................................................... 127
ii
1 Introduction
1.1 Current Neurobiology
The study of the human brain´s function is undoubtedly one of the most fascinating fields of
science today. Finding out why human beings act the way they do has always been a
fundamental question. In the last decades, however, the elucidation of complex
neurobiological processes seems to have come i