Research

Signaling dynamics in live single cells

A fundamental property of living cells is their extraordinary ability to sense and properly respond to a changing environment. Through highly interconnected networks of signaling proteins, internal and external cues are constantly evaluated to adjust cellular behavior. This ability is specially remarkable if one considers that signaling networks operate in the context of high macromolecular crowding, variable protein copy numbers, and are ultimately governed by the stochastic nature of molecular interactions. Through genetic and biochemical approaches coupled with powerful fluorescent biosensors, live cell imaging, and single molecule approaches, we explore and define new biological mechanisms that govern cell signaling.

We use as a model the Mitogen Activated Protein Kinase (MAPK) system, a widely conserved network of kinases that is involved in virtually all aspects of cell biology. Dysregulation of this multilayered network of kinases lies at the core of some of the most devastating human diseases including cancer, autoimmunity, and neurodegeneration. While MAPK cascades are among the most studied signaling pathways, two key gaps in our understanding of the network remain:

The mechanisms of activation of the majority of MAP3Ks is unknown.
How MAPK activities impact so many aspects of biology is unclear.

To fill these knowledge gaps, we combine single cell and single molecule live imaging approaches with gene editing and mouse models to enable rigorous examination of the physiological functions of the MAPK signaling network. We have developed new biosensors and fully automated imaging platforms to monitor signaling dynamics in single cells, unperturbed mouse tissues, and developing embryos. Using these approaches we have uncovered spatial and temporal patterns of signaling events that filter biological noise to effect deterministic outcomes. Our work has opened exciting new directions that suggest that the MAPK network is broadly responsible for monitoring and adjusting the biophysical properties of the cell including macromolecular crowding, phase transitions, and the mechanochemical properties of the cytoskeleton.

Our Focus

Signaling and Cell Cycle Dynamics

Cells live in a constantly changing environment, accordingly signaling networks have evolved to be extremely dynamic. To study signaling dynamics and its role in controlling cell cycle we use live cell imaging of fluorescent biosensors. These technologies provide the spatial and temporal resolutions required to understand how single cells sense and properly respond appropriately to a changing environment.

Our Focus

Single Cell Biosensors

Since the discovery of AvGFP, extraordinary progress has been made to observe molecular events in live cells. However the number of signaling events that can be monitored is still relatively scarce. To address this issue, we use synthetic biology and protein engineering to develop new genetically encoded biosensors. These molecular indicators enable exciting new possibilities to study signaling and cell cycle dynamics in live single cells.

Our Focus

Visualizing Single Molecules in Live Cells

Cell signaling is governed by the stochastic nature of molecular interactions. To understand how cell fate decisions emerge from this stochasticity, approaches that link the behavior of single molecules with cell fate are needed. We combine live super-resolution microscopy of single molecules with single cell biosensors to understand how order emerges from chaos.

Our Focus

Understanding Antigen Discrimination by T Cells

T cells are constantly making life-or-death decisions based on minute differences in antigen, a process known as antigen discrimination. This process relies on the ability of T cells to measure the temporal properties of the TCR-Antigen-MHC interaction. We use a combination of unique mouse models, live cell biosensors, and advanced image analysis tools to study how T cells evaluate antigens in real time.

Our Focus