Megan C. King
Department of Cell Biology
333 Cedar StreetNew Haven, CT 06520
2011 Searle Scholar
Mechanical Coupling of the Nucleus and Cytoplasm
Every cell possesses an integrated mechanical network. Molecules within cells, particularly elements of the cytoskeleton, have the capacity to generate force, drive movement, and thus do work. We are particularly interested in how this mechanical network regulates processes within the nucleus. The nucleus is linked to the cytoskeleton by a complex of membrane proteins embedded in the nuclear envelope called the LINC complex. These molecular bridges provide a means for the cytoskeleton to do work on the nucleus, such as driving nuclear position during cell migration. Our group has found that productive nuclear movement requires attachment of chromatin to the nuclear face of LINC complexes, suggesting that the DNA itself is mechanically important. This observation also begs the question: might these complexes provide a mechanism for signals to be mechanically transduced between the cytoplasm and nucleus? Our lab uses a fission yeast model, Schizosaccharomyces pombe, to further investigate the mechanical functions of the LINC complex within the cell.Chromatin and Nuclear Mechanics: In order to investigate how chromatin contributes to the mechanical behavior of the nucleus, we are establishing methods to apply force spectroscopy to isolated S. pombe nuclei. Using genetic mutants, we can isolate nuclei with altered parameters such as the ratio of chromatin to nuclear volume and the attachment of chromatin to the nuclear envelope. Applying this experimental system, we hope to define how different components of the nucleus define overall nuclear mechanics. Ultimately, we would like to understand how nuclear mechanics contribute to cell behavior, particularly with regards to cell migration.
DNA Repair: In our quest to identify the chromatin regions that are bound to LINC complexes in vivo, and are thus the most likely targets of mechanical forces, we discovered that persistent double-stranded DNA breaks (DSBs) associate with the LINC complex. The cell possesses a variety of mechanisms to repair a DNA lesion, each with a distinct profile of efficiency and risk of mutation. We are currently investigating how the LINC complex and its associated cytoskeletal forces might affect this choice. Using the wealth of genetic mutants in DSB repair and checkpoint proteins available in S. pombe, we are defining how persistent DSBs are recruited to the LINC complex. We are also applying in vivo repair assays to address how association of DSBs with the LINC complex alters repair pathway choice. In particular, we have developed live-cell assays to visualize DNA repair in real time, which allows us to directly investigate the consequence of attaching a DSB to the LINC complex and the cytoplasmic cytoskeleton during
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