As a "cross-disciplinary researcher", I am broadly interested in the engineering of biological systems.  My current research focuses on the structure of the mammalian somatic nucleus, and is primarily in three parts, applying various tools from biophysics, biochemistry and microscopy.

Rheology of the in situ somatic nucleus

I have used a micropipette aspiration creep assay (schematized to the right) to characterize the material properties of nuclei within various living mammalian somatic cells, including epithelium, fibroblasts, and hematopoetic stem cells.  By perturbing the structure of the nucleus in a specific manner, I have been able to determine the relative contribution of chromatin and the Lamin A/C network to nuclear deformability.

The set of images to the left show the qualitatively different behavior of the chromatin and the lamina in a photobleaching assay.  The chromatin flows inside of the nuclear aspiration, exhibiting a viscoelastic/viscoplastic behavior.  The lamina, on the other hand, deforms as a solid-like shell.

It is also possible to make quantitative measurements of nuclear deformation using a creep experiment, and the deformation profiles of various cells or cells exposed to various treatments can be compared.  Using this method, I have demonstrated for the first time that the mechanical properties of the nucleus can change dramatically in cells as a function of their differentiated state.  For example, the nuclei of hematopoetic stem cells are much softer than of differentiated tissue fibroblasts, and the nuclei of human embryonic stem cells stiffen as they are induced to differentiate.  This could potentially be due to many structural changes in the nucleus, for example, changes in heterochromatinization, or expression of nuclear lamins.  I have shown that knocking down Lamin A/C in a human lung cancer epithelium cell line, A549, is sufficient to recapitulate the difference observed.

Finally, large deformations of the nucleus are irreversible, as depicted above left.  In this case, a TC7 epithelial cell has been aspirated for roughly 300s at 1kPa applied pressure (inside of a live cell), and then ejected from the pipette with a gentle back pressure.  The induced deformation is irreversible (a plastic deformation) at least on the order of tens of minutes.

Cysteine Accessibility to Measure Protein Structure in Live Cells

I am currently involved in extending a method developed in our laboratory using cysteine reactive fluorophors to study the structure of proteins inside live cells.  Previous work established the technique as a viable way to examine the unfolding behavior of spectrin in both an in vitro assay as well as in intact Red Blood Cells (RBCs).  See Johnson et al, [science paper] [blood paper].

My interest lies in applying the method to mammalian tissue culture cells to probe protein structure in live cells.  I am currently engaged in developing both a shotgun approach to identify proteins whose conformations are changed by exposing cells to any perturbative condition.  I am also actively working on extending the technique as a way to examine the structure of specific protein domains in live cells.  This is an active area of research, and I will update with results as they are developed.