Single molecule tracking of Myosin VI in the nucleus of living cells and increasing the toolbox for future studies
FacultiesFakultät für Naturwissenschaften
InstitutionsInstitut für Biophysik
Institut für Molekulare Genetik und Zellbiologie
In the past decades, it has been shown that the 3D organization of the chromatin is essential for many biological processes. Chromosome conformation capture methods were able to reveal previously unknown layers of chromatin organizations. These were also shown to dynamically change during the cell cycle or upon different cell stimuli, and they play essential roles in various regulatory mechanisms. Advances in this field led to the identification of different molecular motors, which play specific roles in transcription, gene repositioning, whole chromosome translocation and DNA damage repair. However, most underlying molecular mechanism are still unclear. Single molecule microscopy offers the possibility to study biological systems from a new perspective. I used live cell single particle tracking to study the role of nuclear myosin VI (MVI) in the context of RNA polymerase II-dependent transcription. I found that, besides static chromatin interactions, MVI performs ATPase dependent directed motion. This newly identified process requires the full length motor and might be mediated by cooperating monomeric motors. Additional, actin perturbation and two-color experiments showed that this motion occurs on nuclear actin filaments. In cooperation with the Toseland group chromosome, paint experiments complemented my single molecule studies and showed that MVI is required for transcription-dependent long-range chromatin rearrangements. Overall I propose that the found motion process is directly involved in this chromatin reorganization mechanism. Further studies on this biological system and other nuclear processes will require orthogonal fluorescent labeling systems, and the advances in genome editing present an elegant way to reduce perturbations of the sensitive biological system. I used proteins that are involved in the formation of the transcription preinitiation complex as a model system to establish additional single molecule fluorescent labeling methods, and I added the genome editing system CRISPR/Cas, for direct endogenous labeling of proteins, to the available toolbox.
Subject HeadingsRNS-Polymerase II [GND]
RNA polymerases [LCSH]
Molecular Motor Proteins [MeSH]