We are grateful to the Office of Naval Research, the Army Research Office, the National Science Foundation and the Human Frontiers Science Program for financial support.
Synthetic structural biology
Complex three-dimensional nucleic acid structures can now be programmed to self-assemble with nanometer-scale structural fidelity using base-pair hybridization. This synthetic structural approach offers unprecedented control over the architecture and chemical composition of MegDalton-scale nucleic acid assemblies, including interfacing with natural and synthetic molecules and substrates inside and outside of the cell. However, little is known regarding the design principles required to synthesize functional DNA-based nanostructures successfully. Here, we are developing computational modeling strategies to advance design and assembly techniques based on DNA [3, 4], as well as RNA and synthetic nucleotide analogues. Principal target applications include engineering light-harvesting antennas, multi-enzyme cascades, and cellular imaging probes.
Programming light-harvesting antennas
Natural photosynthetic complexes from purple and green sulfur bacteria consist of highly organized 3D geometric arrangements of chlorophyll molecules to coordinate efficient photon adsorption and transfer for photosynthesis. Programmed self-assembly of DNA into precise 3D architectures can now be used to organize synthetic chromophores and light-harvesting molecules to replicate key aspects of natural photosynthesis . In this work, we are using our computer-aided engineering software design tool CanDo to rationally design optimal light-harvesting assemblies of chromophore molecules using DNA as a programmable structural scaffold . This work is in collaboration with the Yan Lab. Image courtesy Sener et al., PNAS, 2007.
Genome and chromosome architecture
The dynamic, three-dimensional architecture of chromosomes is increasingly being shown to play an important role in mediating gene expression, translocation events, and copy number variation via physical interaction of distant genomic sites that occur due to dense packing of chromatin in the nucleus. Chromosome conformation capture technologies now enable the quantitative measurement of interaction frequencies between millions of distinct genomic sites from bulk cell populations. Fluorescence in situ hybridization further enables the quantitative measurement of chromosome conformation and nuclear positioning at the single-cell level, and fluorescence recovery after photobleaching and fluorescence correlation spectroscopy enable single-cell-level quantitation of chromatin activation state. We are using these datasets to infer the local and global structure and dynamics of chromatin to connect functional genomic measurements with genome conformation. Image courtesy Yaffe & Tanay, Nature Genetics, 2011.
Human islet amyloid polypeptide aggregation
Non-specific peptide-membrane interactions play an important role in numerous amyloid-related pathologies. Human islet amyloid polypeptide (hIAPP) has been suggested to interact with the plasma membrane in forming cytotoxic aggregates and larger-scale fibrils that lead to beta-cell degeneration in the development of Type-II diabetes, but the detailed mechanism of peptide-membrane interaction and subsequent aggregation remains unclear . In this work we are applying our Bayesian inference procedure for Fluorescence Correlation Spectroscopy datasets to characterize the spatial-temporal dynamics of hIAPP aggregation on the plasma membrane using Total Internal Reflection Fluorescence Microscopy . This imaging based approach uniquely enables the characterization of peptide aggregation in live cells, providing in vivo insight into possible mechanisms of cytotoxicity. This work is in collaboration with the Wohland Lab.
Bacterial cell wall growth and growth inhibition
The cell shape and structural rigidity of Bacillus subtilis, a rod-like bacteria, is conferred through its peptidoglycan (PG)-based cell wall, and characterization of the machinery that governs PG synthesis, crosslinking, and eventual cell elongation remains incomplete. Elucidation of the interactions between these peptidoglycan-synthesizing proteins (PGSP) has the potential to reveal new targets for antibiotics and further our understanding of mechanisms of drug resistance in this and other bacterial species . In this work we are studying the PGSP interactome through quantitative time-lapse imaging of fluorescent PGSP fusion proteins. We are applying our Bayesian inference procedures for single-particle trajectory and correlation spectroscopy datasets to infer in vivo interdependencies between synthesizing proteins that are essential to B. subtilis growth and shape maintenance. High-throughput and objective statistical analysis of PGSP motion and molecular interactions will set the groundwork for models of bacterial cell wall formation that will lead to new understanding of the biochemistry and mechanics of peptidoglycans, as well as to potential discovery of previously unknown bacterial susceptibilities to pharmacological cell wall disruption. This work is in collaboration with the Garner Lab.
Signaling localization and cell shape analysis in migration
Cell migration is a critical process in development, wound healing, and immune response, and its pathophysiological dys-regulation is a fundamental hallmark of cancer metastasis. A systems-level characterization of how cells process external cues to initiate local activation of signaling cascades that lead to particular cell shapes and motility responses will enhance our understanding of mechanisms that govern cell migration . In this work we are developing network-based models of cell migration that integrate measurements of spatial and temporal features of signaling pathway activities, cell shape, and migratory response under the influence of physiologically-relevant extracellular perturbations. These models seek to fill two fundamental gaps within the cell migration research field: the low-throughput nature of live cell imaging used to quantify cell motility, and the limited understanding of how molecular components of signaling pathways collectively orchestrate spatiotemporal polarization in cell shape during early stages of migration. This work is in collaboration with the Lauffenburger Lab.
Chromosome transport and segregation
Understanding chromosome segregation during mitosis and, more specifically, the mechanism of attachment between kinetochores and depolymerizing microtubules during anaphase requires detailed analysis of kinetochore protein composition and the associated physical motions of kinetochores and chromosomes. In addition to advancing our understanding of cell division, such insights into kinetochore function may suggest additional targets for anti-mitotic chemotherapy for cancer. In this work, we are addressing this question using single particle tracking of kinetochores during metaphase and anaphase in HeLa cells combined with novel Bayesian inference approaches for particle trajectory analysis to extract the motion states and motion parameters characterizing kinetochore motions in wild type and perturbed systems. We are correlating these motion states and parameters with the molecular state of the kinetochore to determine and characterize the key protein components that control kinetochore segregation. This work is in collaboration with the Cheeseman Lab.
Transmembrane receptor dynamics
Epidermal Growth Factor Receptor (EGFR) is a physiologically central receptor tyrosine kinase that regulates a wide range of biological processes including cell growth, differentiation, and migration. Aberrant activation of EGFR is implicated in the development of many cancers, rendering it a central target for chemotherapeutics. Here, we are applying our recently published Bayesian inference procedures [9,10,11] to infer spatial-temporal activation dynamics of EGFR from Total Internal Reflection Fluorescence Correlation Microscopy (TIR-FCM) and super-resolution-based single-particle tracking in order to interrogate and characterize receptor density and dynamics at thousands of spatial locations in the cell membrane under wild-type, stimulated, and drug-perturbed conditions.
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