title: Dynamics of chromatin structure and nuclear multiprotein complexes investigated by quantitative fluorescence live cell microscopy and computational modeling
creator: Kappel, Norman Constantin
subject: ddc-570
subject: 570 Life sciences
description: Biology has rapidly been transformed into a mainly data-driven, quantitative science. Demands on biological imaging are moving towards quantitative annotations of genes in vivo. In this work I have studied in detail the spatio-temporal distribution and the molecular interaction of protein ensembles as well as of multiprotein aggregates. I have provided the methodology to estimate biophysical parameters such as diffusion coefficients, anomalous diffusion and the free fraction in the binding equilibrium of protein ensembles using fluorescence photobleaching analysis and numcerical modeling and parameter estimation. On the side of protein complexes I have extended existing single particle tracking approaches to allow to automatically detect the exact timing of mobility changes of single particles in live cells. Here, I was able to provide quantitative parameters also on the diffusion coefficient, anomalous diffusion, velocity and chromatin interaction. The nuclear protein ensemble I studied was murine linker histone H1° fused to GFP. I was able to show that diffusion and binding of H1°-GFP to chromatin can be addressed using photobleaching analysis and numcerical modeling. I have thus obtained diffusion coefficients for wild-type H1° and seven point mutants with differential binding affinity ranging from D = 0.01 mm²/s (strongest binder) to D = 0.1 mm²/s (weakest binder). Likewise, I was able to estimate the free fraction to range from = 400 ppm to = 3000 ppm. Exemplary of large multiprotein complexes I chose PML nuclear bodies (PML NBs), named after their constituent promyelotic leukemia protein. I studied in detail their dynamic mobility during early mitosis, ranging from prophase to prometaphase.  A dramatic global increase in PML NB mobility was found during this period with the diffusion coefficient increasing from D = 0.001 mm²/s at interphase to D = 0.005 mm²/s at prophase. Similarly, velocities increased from v = 0.7 mm/min to v = 1.4mm/min and concomittant with a loss in subdiffusive motion. I was able to establish loss of tethering to chromatin as the most likely reason behind this increase as opposed to material flow or chromatin condensation. Lastly, I was also able to relate the timing of the mobility increase to other important cellular events. The increase of PML NB mobility predominantly occured after nuclear entry of cyclin B1, which irreversibly commits the cell to mitosis, and before nuclear envelope breakdown (NEBD).
date: 2009
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/9786/1/Dissertation_Norman_Constantin_Kappel.pdf
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/9786/2/supplementary_materials.zip
identifier: DOI:10.11588/heidok.00009786
identifier: urn:nbn:de:bsz:16-opus-97869
identifier:   Kappel, Norman Constantin  (2009) Dynamics of chromatin structure and nuclear multiprotein complexes investigated by quantitative fluorescence live cell microscopy and computational modeling.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/9786/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng