A brain isn’t built in a day. Then again, it’s never really finished.
During the development of the vertebrate brain, neurons migrate from their birthplace to different final locations. As they migrate, the neurons process a complex web of environmental signals. Deciphering which signals cause which movements and when is an active topic of research. After neurons establish their home address, they can undergo the remarkable process of synaptic plasticity; they can actually send out projections or migrate to make new connections with neighboring neurons.
Scientists in the Cooper Laboratory (Basic Sciences Division) study the signaling pathways that direct neuron migration. Previous research had suggested that an extracellular protein called Reelin, which is secreted by specialized cells, promoted directed neuron migration in developing mammalian brains and also regulated synaptic plasticity in adults. It was found that Reelin binding to membrane receptors on the neuron surface led to clustering of the receptors, phosphorylation of an adaptor protein called Dab1 by Src family kinases (SFKs), and downstream activation of cell migration machinery including the small GTPases Rac1, Cdc42, and RalA. The signaling leads to the directed growth of actin filaments that push the cell forward. While it was known that this happens, it was unknown whether stimulation of this pathway was sufficient to drive cell migration in the absence of other signals.
To address this question, postdoctoral fellow Liang Wang in Dr. Cooper’s Lab developed an approach to stimulate Dab1 clustering, which might mimic its clustering and activation by Reelin-receptor binding. Dr. Cooper explained "We've known for a long time that Dab1 is needed for pretty much all that Reelin is needed for, but there is always the possibility that Reelin sometimes signals without Dab1. Liang came up with a way to activate Dab1 without adding Reelin." The results of Liang and Cooper's investigation were recently published in Scientific Reports.
To conditionally stimulate clustering, Liang genetically fused Dab1 to a small domain of the A. thaliana protein Cryptochrome 2 (Cry2), which very quickly, within seconds, forms dimers and higher-order oligomers when illuminated with blue light. He called this construct Opto-Dab1 and it also included a fluorescent protein, mChFP, so that the protein itself could be visualized.
Stimulation with a focused blue laser induced Opto-Dab1 clustering in mammalian cells grown in culture, as visualized by fluorescent microscopy and TIRF imaging. The clustering was quick (t1/2~50s) and also reversible, with the clustered Dab1 fluorescent signal disappearing from the membranes of cells in the dark (t1/2~10m).
The clustering of Dab1 was known to induce its phosphorylation by SFKs and the specific sites that are phosphorylated have been mapped. Liang found that light-induced clustering of Opto-Dab1 led to phosphorylation of Dab1 and that mutating the known phosphorylation sites in Dab1 blocked this. Therefore, clustering Dab1 is sufficient to cause its phosphorylation. Next, Liang tested whether the subsequent steps of the activation were carried out following clustering of optoDab1 in neurons in the absence of Reelin. In agreement with previous data on endogenous Dab1 activation, he found that clustering Dab1 led to translocation of Akt kinase to the plasma membrane at the site of Dab1 clustering. This translocation was lost when the Opto-Dab1-5F mutant that cannot be phosphorylated was used. Additionally he found that blocking P13 kinase using the drug wortmannin also blocked Akt translocation, as expected. Collectively, their experiments demonstrated that Dab1 clustering led to activation of downstream proteins and re-localization to the site of illumination on the plasma membrane.
This photoactivation method offers finer spatial control of protein clustering than drug induced clustering because drug will diffuse away from the introduction site while the beam of light is kept in a fixed position. Liang used this point to discover how proteins activated by the Dab1 pathway are localized following focused Dab1 activation. He found that activated Akt, and therefore the PIP3 it binds to, could localize up to 10 micrometers away from the site targeted by the 6.6 micrometer diameter laser. In contrast, the downstream protein Crk was localized more closely around the illuminated region.
Excitingly, Liang found that light-induced clustering of Dab1 led to membrane ruffling, lamellipodia and/or filopodia formation near the site of illumination in cells. He even observed directed movement of COS7 monkey cells, NIH3T3 mouse fibroblasts, as well as in primary mouse cortical neurons.
In the future, optogenetic tools such as those developed in this work may help researchers uncover how signaling pathways work in the context of a multicellular living organism such as a mouse.
Wang L, Cooper JA. 2017. Optogenetic control of the Dab1 signaling pathway. Scientific Reports. 7:43760. doi: 10.1038/srep43760.
Funding for this research was provided by the National Institutes of Health.