The Zhang lab recently published a paper in Cell Chemical Biology titled "Optogenetic delineation of receptor tyrosine kinase subcircuits in PC12 cell differentiation."
Neuronal development involves an assortment of growth factors and signal receptors that are activated during different stages. In some cases, a single growth factor can interact with multiple receptors resulting in distinct outcomes such as neuron growth, differentiation, or even death. These complexities make it difficult to study the contributions of growth factors and their corresponding receptors.
The Zhang lab is interested in nerve growth factor, or NGF, which controls neuronal survival and differentiation. NGF binds to two receptors: TrkA and p75NTR. TrkA is considered a high-affinity receptor, while p75NTR is a low-affinity receptor. These two receptors work co-operatively and independently. Additionally, p75NTR binds pro-NGF, a precursor to NGF. This interaction leads to cell death.
How does one delineate the individual functions of TrkA and p75NTR? Using a signaling molecule, or a ligand, would be unhelpful because it would bind to both receptors. Another approach would be to delete one of the receptors and study the downstream effects. This approach is also unhelpful because it creates an on/off situation which is rarely seen in cells.
The cellular environment comprises a dynamic range of signals that are tuned to different concentrations of ligands. To replicate this condition, the Zhang lab has developed an optogenetic system where specific receptors are activated using light. “Although we don’t add any ligand, using this method we modify TrkA to respond to light, thereby allowing us to trace the pathways it controls in the presence of NGF,” said Zhang, Assistant Professor in the Department of Biochemistry.
Other labs reported contradictory results with respect to TrkA function. It was unclear whether Y490 and Y785, two docking sites of TrkA that interact with signaling molecules inside the cell, stimulated or inhibited neuronal differentiation. “We believe that these discrepancies are due to off-target effects of NGF, such as binding with p75NTR,” said Zhang. Using optogenetics, the group showed that both Y490 and Y785 activate cell differentiation in an additive manner.
Optogenetics will help provide further insight into neuronal disorders and cancer, because the contributions of individual proteins can be better understood. Furthermore, optogenetics can be used to study other cellular processes, such as embryonic development, which involve a gradual change in signal intensity. “We can use light to activate a certain molecule. After activation, the light can be turned off, which will lead to a slow decrease in signal intensity. Therefore, optogenetics is a powerful tool in mimicking these subtle modulations,” explains Zhang.
In addition to Prof. Zhang, biochemistry graduate research assistants, John Khamo and Vishnu Krishnamurthy, as well as graduate student Qixin Chen and Prof. Jiajie Diao from the University of Cincinnati College of Medicine, contributed to this work.
Written by Ananya Sen, Graduate Student in Microbiology, Imlay lab.
