Biophysics of Cellular Systems
Dahl - Current Research
For the past dozen years I have led a multidisciplinary research group to study the biophysics of cellular systems. We study mechanics of the nucleus and the actin cytoskeleton to address a wide range of medically-relevant questions. Focusing on our strengths incorporating physics and engineering into complex biological questions has led us to a large range of biomedical applications.
Structure and Mechanics of the Cell Nucleus
It has been more than a decade since the human genome was sequenced, but genetics has not enabled significant advancements in tissue regeneration, cancer, aging, and other diseases. Generally, cellular manipulation and engineering is limited by poor control of genome function. The nucleus of the cell contains its DNA and other gene regulatory factors, and the nucleus is interconnected with the actin cytoskeleton and the genome (Figure 1). My group and I study structure and mechanics of these integrated cellular systems. Our combination of experimental engineering and biology with computation and theory has made us one of the leading authorities in nuclear mechanics and nucleoskeleton structure. For example, with a grant from NSF and a breakthrough biophysical publication, we have identified spectrins as new functional mechanical components of the nucleoskeleton.
Nuclear Rheology and Cellular Phenotype
Cells respond to a variety of chemical and mechanical signals. Mechanobiology is the study of the propagation of mechanical cues within the cell; applied force, altered mechanical environment and intracellular forcegeneration all impact cell function. Mechanical forces outside the cell including shear stress and compression can propagate into the nucleus and alter sub-nuclear structures and movements. Using live-cell imaging and particle-tracking algorithms, particle velocimetry and rheological analysis, we observe distinct regimes of cellular responsiveness to force (Figure 2). Stimulation from chemical factors increase cellular force generation, which impacts nuclear changes. This work has been funded by an NSF CAREER
award, and we have used techniques developed to address numerous disease states including premature aging, vascular dysfunction and wound healing. We have published the seminal works on the mechanical and structural aspects of lamin A Hutchinson Gilford progeria syndrome in the nucleus, and neural leukodystrophy.
Advanced Imaging Technology and Chromatin Organization
Using careful controls we have been able to use particle tracking imaging to measure levels of chromatin compaction in live cells and correlate levels of functional heterochromatin with mechanical properties in live, unperturbed cells. We have also correlated these changes in effective diffusivity within the entangled polymeric network of the nucleus to changes in fluorescence lifetime imaging (Figure 3). These complementary techniques –one pro viding a single snapshot of fixed cells with spatial resolution and another providing information regarding force generation and chromatin condensation –allow a full characterization of the rheological properties of the nuclear interior. The work has been sponsored by the NSF. We are currently using these techniques to measure the pathways of DNA damage repair in normal cells. Also, we are quantifying DNA repair in different breast cancer cell lines in response to chemotherapeutic agents to determine what makes certain breast cancer cells resistant to drug treatments. Similarly, we are using measurements of chromatin condensation to examine nuclear changes associated with cellular infection by Herpes Simplex Virus 1 as a model for DNA viruses.
Protein Engineering and Thermodynamics
The study of protein structure, folding, stability and thermodynamics includes developing the tools to produce and characterize pure structures. We have also mastered proper biophysical characterization and spectroscopy, simulation to complement the assays as well as asking important questions with the proteins of interest (Figure 4). We have developed the experimental techniques and established collaborations to address aspects of protein stability, protein-ion interactions, protein-membrane binding, intrinsically disordered structures, post-translational modifications of proteins and protein-nanomaterial interactions. These studies were funded by the Progeria Research Foundation and an NIH F31.
Single Wall Carbon Nanotubes (SWCNTs) as Subcellular Biomaterials
In collaboration with Mohammad Islam (Material Science and Engineering at Carnegie Mellon) we are developing purified and dispersed single wall carbon nanotubes (SWCNTs) as subcellular biomaterials. We quantify surface interactions to improve dispersion of SWCNTs, study cellular uptake and utilize unique spectral properties of the SWCNTs to examine features inside cells. Funded by an NSF NER, this work has generated 16 publications.
Dispersion of SWCNTs with Proteins
By dispersing short SWCNTs (150nm) with proteins inclusing bovine serum albumin (Figure 5), we are able to increase delivery to cells to 107 SWCNT per cell without deleterious effects. This is orders of magnitude above other delivery systems reported. We examine uptake into many model cell lines including macrophages for future use in subcellular sensing, tracking in situ, drug delivery, thermal ablation and subcellular actuation. We have also used the drug binding pocket of albumin for drug delivery, and we are using engineered proteins (Figure 4) for targeted delivery to cellular compartments.
SWCNT Interactions with Subcellular Structures
Through a combination of cellular and purified lipid membrane studies, we have proven that SWCNTs enter cells via active endocytosis and not membrane penetration. SWCNTs coated with PF127 enter the cytoplasm of cells and then associate with the F-actin cytoskeleton (Figure 6). SWCNTs are not acutely toxic, but at extremely high levels, the SWCNT-actin association can inhibit cell division, reduce cell force generation and generally reorder structures. Conversely, SWCNTs coated with bovine serum albumin, remain within the metabolic pathway. This specified subcellular delivery will allow for nanoscale cellular modulation.