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Bioenergy & Defense

Bioenergy & Defense Technologies

Research: Biodefense


Biological Imaging - Pathogen Detection Tools - Protein Characterization


Biological Imaging

Superresolution Imaging of Innate Immune Response

The development of optical approaches with effective resolutions below the diffraction limit promises to revolutionize the field of biological imaging.  We have implemented one such method – Stochastical Optical Reconstruction Microscopy (STORM) for use in studying innate immune signaling.  We’ve observed unique TLR4 receptor behavior that may help explain why certain pathogens such as E. coli produce a robust immune response, while others such as Y. pestis (plague) do not, due to the increase in available resolution – down to <50nm. 

Raman imaging of Haematococcus pluvialis

STORM imaging of TLR4 and lipopolysaccharide distribution in mouse macrophage cells (Left - E. coli LPS; Right = Y. pestis LPS). Images reveal subtle changes in receptor clustering in the cell membrane in response to different antigens that are not apparent in conventional resolution microscopy.
   

Cell-Nanoparticle Interactions

Although nanomaterials are rapidly being incorporated into an increasing number of consumer products, fundamental knowledge about their interactions with living systems is limited.  We have used advanced imaging approaches to understand the role particle shape and size play in determining particle behavior on the cell membrane, as well as their ultimate fate within the cells’ lysosomal machinery.  In particular, we observed that particles with higher aspect ratio diffuse much more slowly in cell membranes, and are internalized at a lower rate. 

MCR analysis of LHCII and PSII in Chlamydomonas reinhardtii

Hyperspectral imaging of CdSe quantum dots in living mast cells reveals differential membrane partitioning and lysosomal sorting based on particle size and shape.


TIRF Microscopy of Mast Cell Signaling

TIRF microscopy offers an ideal method for interrogating interfacial biological phenomena.  We are studying the interplay between the IgE signaling pathway and mast cell degranulation, which both occur at the interface between the cell membrane and an external immuno-stimulatory environment.  We are currently using RBL cells transfected with a GFP-tagged exocytosis marker (FasL) atop an antigen-containing supported lipid bilayer in order to develop a single cell degranulation assay, and to conduct fundamental research into this process.

Dual-color TIRF microscopy of RBL-2H3 cells

Dual-color TIRF microscopy of RBL-2H3 cells containing a GFP-tagged exocytosis marker (FasL, in green) and FceRII receptors (red) show the interplay between IgE signaling and mast cell degranulation.


Resolving Dynamics of Cell Signaling via Real-Time Imaging of the Immunological Synapse

Real-time image of immunological synapse
Cellular response is determined by dynamic, stepwise interactions of receptor proteins with key membrane proteins. We determined that receptor proteins and signalling is corralled by intracellular cytoskeletal structures.


Virulence Membrane Protein Organization and Complex Formation in Francisella Novicida

Pathogenesis depends on molecular interactions between immune cells and bacteria/viruses. Here, we see virulence proteins accumulate and attack cells only 40 minutes after infection.
      Image of virulence proteins in Francisella Novicida


Visualizing Cholesterol Dynamics in the Living Cell Membrane

Cholesterol adds membrane firmness and maintains fluidity. In response to ligand binding induced signal transduction, cholesterol forms a punctate structure to regulate membrane properties.
  Image of cholesterol in the cell membrane of a resting cell
Resting
Image of cholesterol in the membrane of an activated cell
Activated


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Pathogen Detection Tools

Pathogen-Driven Cytokine Regulation in Isolated Individual Cells


Single Cell Array Chip Single Cell Array Chip Diagram

Immune response is governed by pathogen detection and cell to cell communication. Pathogen detection initiates a primary signal while subsequent cell-cell communication represents a secondary signal. In mixed populations, it is impossible to separate primary from secondary signals. We use microfluidics, imaging, and genetic tools to hydrodynamically isolate and interrogate cells. In this case, we observe the translocation of a transcription factor (RelA, green) into the macrophage nucleus, initiating gene transcription as a response to the primary signal only (bacteria in red). In this way, we measure the kinetics and characterize networks of complex interdependent signal transduction pathways.

Diagram of host/pathogen interaction
Translocation of transcription factor RelA into macrophage nucleus


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Protein Characterization

Resolving the Conformation of HIV Nef Bound to Lipid Membranes by Neutron Reflection


Neutron reflection of HIV Nef bound to lipid membranes

Nef is one of six HIV-1 accessory proteins and directly contributes to AIDS progression. Nef associates with membranes and may require a transition from a solution conformation to a membrane-associated conformation. It has been hypothesized that a transition in conformation enables interaction of Nef with cellular proteins. Despite its obvious diseases importance, there is little or no direct information about the conformation of membrane-bound Nef. In this work, we are using neutron reflection to resolve the conformation of membrane-bound Nef and TIRF to study its interaction with cellular proteins.


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