Signaling dynamics: coded messages for cell fate choice

Communicating information across spatio-temporal scales is a problem central to all multicellular life. Biochemical events like protein phosphorylation occur on a timescale of minutes, yet phospho-signaling regulates cell fate transitions over days and maintains tissue growth for weeks. How does biology accurately bridge these scales, accomplishing developmental transitions at just the times and locations that they are needed?

Although this central question in cell signaling is still wide open, live biosensors have opened the door to a new, more precise picture of tissue-scale signaling. These biosensors have revealed that pathways often do not just turn from OFF to ON upon stimulation. Instead, they have uncovered an entire missing language of cell signaling – pulses and traveling waves of protein activity – that may carry information about past stimuli and future decisions. Some of the most profound examples involve oscillating signals, including traveling waves across the skin during wound healing, to pulses of endocrine hormones throughout the body. It is our goal to understand how signaling pathways dictate fates. In order to accomplish this we will need to both “listen” to this language of dynamic signaling in vivo, and “speak” it by delivering dynamic stimuli to just the cells and tissues of our choice using optogenetics.

Erk dynamics in a developing fly embryo

Drosophila melanogaster (fruit fly) is our primary model system in which we explore developmental decision-making at the tissue, organ, and organism scales. It is ideal model system for our work as Drosophila larvae and embryos undergo rapid growth and maturation in a matter of a few days, they are transparent (allowing for the passage of stimulating and imaging light), and they are readily accessible to genetic manipulation. Thus they offer a unique opportunity for us to study signaling and dynamics in the context of real and complex biology that has not been visualized previously. In addition to fruit flies, we also utilize mammalian cell culture to get a closer look at subcellular dynamics and develop new tools.

Developing optogenetic tools to control and probe signaling biology

Using the roadmap provided by biochemistry and genetics we can develop optogenetic various optogenetic tools to control signaling at specific nodes in a pathway. Optogenetics provides precise spatiotemporal manipulation, allowing for near instantaneous and controlled activation of signaling pathways. When combined with classical genetic perturbations, light can become the sole source of activating signal, giving complete control over the pathway. This allows one to test exact signaling requirements and manipulate dynamics and patterns

wild type

genetic LOF

LOF + opto rescue

intensity

Understanding the role of signaling in homeostasis and disease

Even once development is complete, our body must still constantly maintain and repair itself. Signaling pathways play a critical role here in instructing cells to perform these behaviors. Ectopic or misrelated signaling often leads to disease states. Understand how and why this happens is fundamental to treatment and prevention of many diseases.

normal signaling

correct fate

misspecified fate

disease signaling

fate A

fate B

ectopic Erk signaling

Signaling integration of mechanical and biochemical cues.

Signaling can originate from the substrate on which cells crawl and is dependent on extracellular matrix (ECM) type, density, and stiffness. As cells crawl along substrates they often produce numerous filopodia - small, finger-like protrusions made up of bundled actin filaments. Recently, it is becoming appreciated that filopodia can serve as both mechanical and chemical sensors of the microenviroment.

PI3K signaling (Akt-PH; hot colors) at the sights of focal adhesion formation (Paxillin) where filopodia were present

PI3K signaling (hot colors) along filopodia in response to nearby induction of protrusion by photo-activation of Rac

Filopodia have primed integrins localized to their tips and are enriched for several signaling components. These integrins, the receptors which physically link the cell with the ECM, can transmit both mechanical and chemical signals to the cell. Integrins can engage with specific motifs in the ECM, transmit forces to the cell, and eventually form focal adhesion complexes which recruit a number of signaling and cytoskeletal components to the site. The mechanisms of mechanosensing and for how signals are transmitted for different forces, substates, and integrins remains unclear. We seek to delineate these mechanisms utilizing high-resolution Total Internal Reflection Fluorescence (TIRF) microscopy and optogenetic tools.