Probing intercellular signaling at the nano scale level
Our main research goal is to understanding how cells coordinate their differentiation in space and time during embryonic development to form complex patterns of differentiation. To perform these patterning processes cells use extracellular signaling to communicate with their neighbors as well as intracellular genetic circuitry to interpret signaling and make cell fate decisions. We are focusing on developmental patterning processes in which neighboring cells adopt different fates (such as differentiation of neural precursors into neurons and glia). In metazoans, the canonical signaling pathway that coordinates such processes is the Notch signaling pathway. Notch signaling pathway is used for transferring information between neighboring cell: Delta ligands in one cell can interact and activate Notch receptors in a neighboring cell. Notch signaling misregulation often leads to disease states such as cancer and developmental disorders.
While we know a lot about the molecular mechanisms of the Notch signaling pathway, we have very little understanding of how the Notch and Delta find each other across the boundary between cells, whether Notch and Delta are evenly distributed or localized in subdomains at the membrane, and how the properties of boundary between the cells affect signaling. To address these questions we need to develop tools that would allow us to measure the dynamic distribution of Notch receptors and Delta ligands on the plasma membrane, to characterize and control the properties of the boundary between cells, and to quantitatively measure the interaction between Notch and Delta across cellular boundaries.
Our effort in these directions will focus on the following goals:
Mapping the spatial distribution of Notch and Delta on the membrane and on the boundary between cells. The spatial distribution and dynamics of the Notch and Delta proteins on the cell membrane is largely unknown. Several tools may allow us to image these distributions. First, we would like to develop an AFM based technique in which Delta (Notch) can be attached to the AFM tip and would probe Notch (Delta) distribution on membrane plasma. A second approach would be to label Delta or Notch with photoactivatable fluorescent protein and use super resolution techniques such as PALM to map the spatial distribution of Notch and Delta in high resolution. In particular it would be interesting to follow the dynamics of Notch and Delta at the plasma membrane and in the boundary between neighboring cells.
Develop tools to characterize and control the boundary between cells. How cell-cell signaling is affected by the geometrical, mechanical, and molecular properties of the boundary between cells? To address this question we will develop a set of tools that would allow characterization of the boundary between cells using super resolution imaging techniques, control of geometry of the boundary using surface patterning, and control of the molecular properties of the boundary by controlling cell morphology regulators. We will also use nano-fabrication techniques to measure and apply forces on the boundary between cells and monitor its affect of intercellular signaling. These tools will be combined with current and future tools to monitor intercellular signaling.
Quantitative measurement of Notch and Delta interactions across cell boundary. While techniques to track proteins at the cell membrane are currently available, we do not currently have ways to measure the interaction between proteins across cell boundary. We therefore propose to develop such molecular tools such as fluorescent complementation assay to study the interaction between Notch and Delta across cell boundaries.
The combination of these techniques will provide a powerful set of tools for understanding the interplay between intercellular signaling and cell morphology.
Visualizing dynamics of Notch signaling in single cells. Quantitative time lapse micrscopy of CHO cells expressing Notch receptors responding to trans-Delat (Delta plated on the cells) and to cis Delta, expressed in the same cell (Red fluorescence). Notch reporter (green fluorescence) turns on when the level of cis-Delta goes below a threshold. Analysis of such movies allows the characterization of the input-output response function of the Notch signaling pathway. This analysis showed that cells can either be 'senders' or 'receivers' but not both.
Simulations of developmental patterning processes. (Top) A model for generating lateral inhibition patterns from an intially uniform field of cells. Neighboring cells inhibit each other's Delta activity the Notch signaling. The resulting multicellular feedback can generate an alternating patterns of differentiation (bottom) where one 'high Delta' cell suppresses its neighbors. The process is studied through analytical methods and numerical simulations.