Nan Laboratory Research Overview

Research in the Nan Lab focuses on nanoscale spatial systems biology and specifically on understanding how oncogenic signaling modules assemble into functional complexes in cells and operate in their native cellular context. We also seek to translate such mechanistic understanding into novel cancer therapeutics. To achieve these goals, we take a multidisciplinary approach that combines biological nanoscopy, biochemistry and bioengineering, and computation in our research.

Biological nanoscopy for quantitative analysis of cell signaling architectures

We develop and apply superresolution fluorescence microscopy (SRM) techniques for quantitative assessment of the spatial organization and molecular composition of signaling architectures. SRM techniques such as photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and their derivatives break the diffraction limit of conventional light microscopy and enables cellular imaging with nanometer spatial resolution and single molecule sensitivity (Fig. 1).

Figure 1: PALM/STORM graphics (left); PALM image of PAmCherry1-tubulin in U2OS cells (right).

Figure 1. Single molecule superresolution microscopy.

We have demonstrated the use of quantitative SRM for stoichiometric analysis of protein oligomers (Nan et al. PNAS 2013; see also Fig. 2, top panel). More recently, we have shown that by combining bimolecular fluorescence complementation (BiFC) with PALM, we can localize and track protein-protein interactions with nanometer spatial resolution and single molecule sensitivity (Nickerson et al. PLoS ONE, 2014; see also Fig. 2, middle panel). Lastly, correlative electron and superresolution microscopy allows detailed investigation of both the proteins and protein complexes of interest and their cellular context at the nanoscale (Fig. 2, bottom panel). More technical developments to enhance the capability of SRM techniques in studying complex signaling pathways and probing protein-protein interactions are currently underway.

Figure 2: Imaging protein dimers directly with quantitative PALM (top); BiFC-PALM for visualizing protein-protein interactions with nanometer resolution (middle); Correlative superresolution and electron microscopy (bottom)

Figure 2. Superresolution and correlative microscopies for quantitative biological imaging.

Spatial orientation and biological functions of membrane clusters of ErbB receptors and Ras GTPases

Another main focus of our research is to use the tools developed above to understand the spatial orientation and biological functions of ErbB receptors and Ras GTPases in the context of human cancers. Both ErbB receptors and Ras GTPases are master regulators of cell signaling (Fig. 3, left). Activated ErbB and/or Ras signaling are among the most common molecular drivers in human cancers, but therapeutic targeting of these molecules remains a challenge. Accumulating evidence indicate that the spatial orientation of these molecules, for example formation of membrane clusters, is critical to their biological activities (Fig. 3, right).

Figure 3: Schematic of ErbB and Ras signaling structures on the cell membrane (leftt); Immuno-EM and PALM/STORM micrographs (right). CLICK to view full-size version.

Figure 3. ErbB and Ras signaling involves formation of higher order structures (click to view full size).

We combine quantitative SRM with biochemistry and computation to understand how ErbB and Ras signaling structures form on the cell membrane and how their spatial orientation may impact the signaling outcome under physiologic, pathologic, and pharmacologic conditions (Fig 3, right). This approach was previously used to visualize Raf dimers and multimers that form under various activation conditions, for example in the presence of mutant Ras or Raf inhibitors (Fig. 4). Similar analysis on ErbB and Ras is ongoing.

Figure 4: Schematic of cytosolic RAF monomers dimerized by Active Ras vs. dimerized by RAF inhibitors (left); PAmCherry1-CRAF dimer micrographs (top right); bar graph plots of Molecule Density vs. Dimerization of CRAF (right bottom)

Figure 4. Direct visualization of Raf dimers and multimers in cells with PALM (click to view full-size).

Research Sponsors

Research in the Nan Lab is funded by these sponsors:
Knight Cancer Institute; Damon Runyon Cancer Research Foundation; FEI; National Cancer Institute, and the M.J. Murdock Charitable Trust.

Sponsors of the Nan Lab: M.J. Murdock Charitable Trust, Knight Cancer Institute, FEI, National Cancer Institute, Damon Runyon Cancer Research Foundation.