We seek to understand how human cells preserve their genome integrity
Single Molecule Live Cell Imaging
SEEING IS BELIEVING...
Single molecule imaging is a powerful technique. It allows the observer to measure the behavior of individual molecules rather than the average of the whole population. For instance, it revealed to us that telomerase in the movie above in green, behaves differently at telomeres (red) and Cajal bodies (blue) than at other nuclear locations. To visualize single proteins in living human cells we take advantage of the HaloTag, a small protein, that can be covalently modified with a cell permeable small molecule fluorophore. We expose cells expressing a HaloTagged protein from its endogenous locus to a small dose of fluorophore for a short time to label only a subset of the molecules in the cell. Using this approach we achieve a particle density that allows the detection of single molecules, without much overlap between the fluorescence signals. We visualize the fluorescent molecules using a TIRF microscope equipped with powerful lasers and EMCCD cameras and we can image multiple channels with high temporal resolution (50 frames per second). To aid the detection of single molecules we generate a thin light sheet through the sample, to reduce out of focus fluorescence, a technique called Highly Inclined Laminated Optical Sheet (HILO) microscopy. If you are interested in learning more about this technology, or are potentially interested in setting up a collaboration, please contact Dr. Schmidt.
CRISPR/CAS9 MEDIATED GENOME EDITING...
... is a powerful technique that allows us to make precise changes in the genome of human cells. Until recently fluorescently tagged proteins were usually expressed by retroviral transduction or transient transfection of plasmid DNA, which frequently leads to over-expression on top of the endogenous protein that the cell is already producing. We use a two-step genome editing strategy to introduce fluorescent tags at the endogenous loci of the proteins that we are interested in studying. In addition to the fluorescent protein, we also include an affinity tag to allow purification of the protein of interest. By modifying the endogenous locus we can achieve expression levels that are similar to the wild type protein and any regulation of the expression of the protein of interest is unchanged since all the regulatory elements around the gene are identical. This is especially important for imaging the trafficking and localization of proteins in the cell, since over-expression often alters the cellular distribution of the respective protein.
In addition to introducing fluorescent tags into the human genome we also use CRISPR/Cas9 technology to knock out target genes or to make specific changes to existing genes, such as point mutations and truncations.