% This functions simulates diffusion together with binding kinetics
% The initial Free concentration can be optionally supplied
% Returned is the FRAP recovery curve inside the bleach-ROI
%
% Synopsis: [curve, movie] = simBindingDiff(NucRadius, SpotsRadius, XDistance, Spot1Int, Spot2Int, Koff)
% Spot1Int, Spot2Int are the concentration of binding sites!
function [Cfree, Cbound, CFRAP, BoundToFreeRatioInBox, CbFit, HalfBound, movFree, movCb] = simBindingDiffFixedComp(NucRadius, SpotsRadius,
FRAPBoxSize, XDistance, Spot1Int, Spot2Int, koff, numsteps, D, pixsize)
% The meaning of Spot1Int is now kon*Ps*cs
if nargin < 9 D = 0.5 % um^2/s
% koff is given in [1/s]
end
if nargin < 10
pixsize = 1.0; % um/pixel end
Dpix = D / (pixsize^2); % in pixel^2/s Koff = koff/Dpix; % in [1 / pixel^2]
dt = 1/Dpix/1.0 % s per simstep / 4.0
[Cfree, Cbound, CFRAP, movFree, movCb, BoundToFreeRatioInBox, BleachedFrac]
=simBindingDiff(NucRadius,SpotsRadius,FRAPBoxSize,XDistance,Spot1Int/Dpix , Spot2Int/Dpix, Koff, numsteps);
BoundToFreeRatioInBox = BoundToFreeRatioInBox*pixsize^2 % convert to um^2
HalfFree=EstimatePos(Cfree,0.5)*dt HalfBound=EstimatePos(Cbound,0.5)*dt
% Fit the free compartment
%FRAPfun = inline('c(1)+(1-(1-c(1))*exp(-t.^0.5./(c(2).^0.5 / log(2))))','c','t');
%FRAPfun = inline('b(1)+b(2)*t./(b(3)+t)','b','t');
FRAPfun = inline('b(1)+b(2)*t./(5+t)+b(3)*(1-exp(-t./(b(4) / log(2))))','b','t');
FRAPstart = [0 0.1 0.3 50];
%freefun = inline('(t./(b(1)+t))','b','t');
%freestart = 0.2;
freefun = inline('b(1).*(t./(b(2)+t))+b(3).*(t./(b(4)+t))','b','t');
freestart = [0.5*100 56 0.52*100 1.5];
%freefun = inline('2/pi.*atan(b(1).*t.^b(2))','b','t');
%freestart = [10.0 1.0];
%freefun = inline('(t.^abs(b(2)))./(b(1)+t.^abs(b(2)))','b','t');
%freestart = [0.2 1.0];
%freefun = inline('b(1)+b(2)*(1.0-exp(-(t.^abs(b(3)))/b(4)))','b','t');
%freestart = [0.001 1 0.5 10];
Cbfun = inline('c(1)-c(2)*exp(-t./c(3))','c','t');
% Cbstart = [12*100 12*100 75];
Cbstart = [3 3 3];
% These are the "measured" values Cfm =
double(squeeze(movFree(floor(size(movFree,1)/2),floor(size(movFree,2)/2), :))); % These are the "measured" values
PreBleachCb = Spot1Int / Dpix / Koff;
MHalfFree=EstimatePos(Cfm,BleachedFrac/2.0)*dt MHalfBound=EstimatePos(Cbm,PreBleachCb/2.0)*dt x=[0:numsteps].*dt;
y=squeeze(double(Cfm)); % Cfree FreeFit=nlinfit(x,y,freefun,freestart)
% Fit the bound compartment
y=squeeze(double(Cbm)); % Cbound CbFit=nlinfit(x,y,Cbfun,Cbstart)
figure
%ba_eq = CbFit(1) % Cb at equilibrium
%bf_eq = FreeFit(1)+FreeFit(3) % Cf at equilibrium
Ibn=Cbm(1:numsteps-2);
VIII ACKNOWLEDGEMENTS
My sincere gratitude and thanks go to Dr. Donna Arndt-Jovin for her ongoing support, encouragement and patience during this time. I am thankful for her care, invaluable insight and criticism and for all the discussions we had. My gratitude also goes to Thomas Jovin for giving me the chance to work in his department where I had the possibility to interact with people from so many disciplines.
I also thank the thesis committee members Prof. Herbert Jäckle and Prof. Hans-Joachim Fritz for discussions and suggestions.
Special thanks are addressed to Greco Hernandez and Cornelia Fritsch for their help and guidance in all the “fly work” and other technical assistance they gave. Thanks for Dr. Peter Schwartz for acquiring electron micrograph pictures and people from Prof.
Jäckle’s lab and Dr. Jürg Müller’s lab for materials and collaboration. Thanks Bernd Rieger for “brainstorming” for the FRAP kinetic model and simulations.
Of course, all the people in the lab are responsible for the great scientific and social environment that is so important for me. All the evening meetings, game nights, parties, were so great with you. I thank Yvonne Fondufe for her friendship, Wolfgang, Peter and Avishay for their funny jokes and nice characters, Diane for initiating the travels with
“Bahnvergnügen” and all the other people, this would be a long list.
I thank the PhD Program in Molecular Biology -International Max Planck Research School at the Georg August University for the great organization and especially Steffen Burckhardt for all he has done for the students and for the success of the program.
I am forever grateful to Rainer Heintzmann for the great scientific experience I had with you, for many hours of discussions, for supporting me in my ups and downs and more than that I am grateful for your love.
IX LIST OF FIGURES
Figure 1 The cell nucleus: organization of the nucleus into specialized domains...- 2 -
Figure 2. Pattern formation in Drosophila.. ...- 5 -
Figure 3. Examples of homeotic mulations. ...- 6 -
Figure 4. Mechanistic requirements for Polycomb group (PcG) silencing.. ...- 8 -
Figure 5. Interactions among Polycomb group (PcG) proteins...- 10 -
Figure 6. Fluorescence recovery after photobleaching (FRAP). ...- 33 -
Figure 7. Inverse FRAP (iFRAP) and three dimensional iFRAP (3D-iFRAP)...- 34 -
Figure 8. Fluorescence Loss In Photobleaching (FLIP). ...- 35 -
Figure 9. Half-FRAP. ...- 35 -
Figure 10. Constructs used to generate transgenic fly lines expressing PcG proteins fused to GFP...- 44 -
Figure 11. Distribution patterns of PhGFP and PcGFP in the embryos and larval wing discs...- 47 -
Figure 12. Chromatin dynamics in diploid nuclei ...- 50 -
Figure 13. Chromatin movement in various regions of the wing imaginal discs...- 52 -
Figure 14. Diffusion of PcGFP in nuclei of preblastoderm embryos...- 54 -
Figure 15. Gamma correction for boundary conditions and box shape...- 57 -
Figure 16. Classical photobleaching techniques in Drosophila embr. and tissues...- 59 -
Figure 17. Secondary bleaching. ...- 60 -
Figure 18. Simulation of the out-of-focus bleaching...- 61 -
Figure 19. Average intensity calculation in a three dimensional weighted volume.- 63 - Figure 20. 3D-iFRAP...- 64 -
Figure 21. Dissociation rate constants for PhGFP and PcGFP ...- 66 -
Figure 22. Confocal images of PhGFP and PcGFP expr. salivary gland nuclei ...- 67 -
Figure 23. Kinetic modeling of PcG binding to PREs ...- 68 -
Figure 24. Bratio.. ...- 70 -
Figure 25. Simulation of the binding kinetics of proteins targeted to localized sites in the nucleus ...- 72 -
Figure 26 Linear dependence of the recovery time t0 on the Bratio...- 73 -
Figure 27. Binding processes slow down the FRAP recovery profile for the free
diffusion molecules ...- 75 -
Figure 28. Distribution PhGFP and PcGFP in the larval salivary gland nuclei and FRAP curves for individual bands...- 77 -
Figure 29. FRAP on the same PcG locus in the salivary gland ...- 78 -
Figure 30. Segmentation of the bound fraction and fitting of the FRAP curves ...- 79 -
Figure 31. FRAP on bound PcG proteins in the salivary gland nuclei...- 80 -
Figure 32. Dissociation rate constants for PhGFP and PcGFP and reassociation rate constants for PhGFP ...- 81 -
Figure 33. Polyhomeotic protein binding to chromatin requires the presence of the Polycomb protein ...- 82 -
Figure 34. Effects of PhGFP overexpression in the fly eye and wing ...- 85 -
Figure 35. Generation of the GFP-ESC construct...- 87 -
Figure 36. Repeated transient interactions during gene activation ...- 88 -
X LIST OF TABLES
Table 1 Polycomb group proteins and their function ...- 9 -
Table 2. Primary antibodies ...- 25 -
Table 3. Quantification of western blots in Fig. 2 G, J and K ...- 48 -
Table 4. Diffusion coefficients for PhGFP and PcGFP ...- 54 -
Table 5. Parameters for a simulated FRAP ...- 71 -
Table 6. ph null phenotype rescue statistics ...- 83 -
Implications for gene expresion" on my own and with no other sources and aids than quoted.
Mathematical modeling of the binding kinetic reactions was done in collaboration with Dr.
Rainer Heintzmann and Dr. Bernd Rieger. The simulation programs used in this work have been written by Dr. Rainer Heintzmann.
exchange rapidly in living Drosophila” Development (accepted)
Ficz .G, Heintzmann, R., Rieger B., Jovin T., Arndt-Jovin D., (2005) “Analysis of binding reactions of localized chromatin binding proteins by Fluorescence Recovery After Photobleaching” (manuscript in preparation)
Master´s Thesis
Gabriella Ficz
“Comparison of diffusion and dissociation constants for proteins in complexes, which modulate transcription in Drosophila: the Polycomb group repression complex”
(International Max-Planck Research School Molecular Biology, 2002)
Oral presentation at the symposium: “Chromatin: Assembly and Inheritance of functional states” in Muenchen, Germany, Oct. 9-11, 2002, entitled: “Mobility of proteins of the Polycomb-Group (Pc-G) and their stability in repression complexes during Drosophila melanogaster development”
Poster presentation at the following meetings:“Dynamic organization of nuclear function” meeting at the Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, Sept. 18- Sept. 22, 2002; FEBS Advanced Workshop: “Nuclear architecture, Chromatin Structure and Gene Control” Wageningen, Netherlands 14-17 November 2003; 46th Symposium of the Society of Histochemistry: “The Cell Nucleus” Prague, Czech Republic 22-25 September 2004; 2nd Alan Wolffe EMBO Conference on Chromatin and Epigenetics, EMBL Heidelberg, Germany, May 19-22, 2005
Name
Date of birth
Gabriella Ficz June 30
th,1978
Education Theoretical Lyceum “Octavian Goga”,
Miercurea-Ciuc, Romania
University of “Alexantru I. Cuza”, Iasi, Romania
International MSc./PhD Program in Molecular Biology, Goettingen, Germany
Max-Planck-Institute for biophysical Chemistry, Department for
Molecular Biology, Göttingen Germany
M.Sc. Molecular Biology
submission of PhD Thesis
1992-1996
1996-2000
2000-present
2001-present
June 2002
June 2005