Manipulating subcellular protein localization in vivo using light
Posted on 14/01/2016
Manipulating the sub cellular location of polarity protein Pard3 in vivo
Researchers from the Department of Developmental Neurobiology at the Institute of Psychiatry, Psychology & Neuroscience (IoPPN), King's College London, in collaboration with scientists from the University of California, San Francisco, have developed an optogenetic method that, for the first time, can rapidly and reversibly manipulate protein localisation in individually targeted cells in living vertebrate embryos. The results have recently been published in the journal ‘Developmental Cell’.
Proteins are involved in virtually all cell functions, and many proteins occupy distinct locations within cells to execute their function. While the inactivation of specific genes or the expression of a specific protein in a tissue in which it is normally not expressed can reveal the involvement of proteins in biological processes, more subtle manipulation of proteins is needed to interrogate their precise roles. For example, to understand how the asymmetric organization of the cell membrane, cytoskeleton and intracellular organelles are established and why the asymmetries are important, it would be helpful to manipulate protein localization within cells.
Over the last decade, optogenetic experimental approaches, i.e. the use of light to control the activity of cellular proteins, have had widespread success in cell culture and in single-celled organisms. However, there is a need to adapt current technologies to multicellular organisms such as vertebrate embryos.
In this new study, the researchers succeeded in adapting a specific light-controlled protein system so that it would be expressed in living zebrafish embryos. Moreover, the team showed that this system is an efficient method to accurately, rapidly, and reversibly recruit proteins to specific subcellular regions within a vertebrate embryo. Lead author Clare Buckley, who worked with collaborator Orion Weiner in UCSF, said "The development of this technique has the potential to change the way we approach biological questions in vivo by allowing us to directly target individual components of a pathway without globally removing them. To be able to do this inside individual cells within the brain of a living fish embryo is pretty exciting." As a proof of principle, the researchers then used the light-controlled protein system to spatiotemporally manipulate the localization of a polarity protein and found that it could rapidly, and reversibly recruit proteins to specific subcellular regions of the embryo. "We’re particularly excited to be able to manipulate polarity protein location within neural progenitor cells. This will help us to understand how the fundamental organisation of the vertebrate brain neuroepithelium is established", said Jon Clarke, senior co-author of the study.
The optimizations of the optogenetic system used and general methodology developed in this study can potentially enable the use of this system to reversibly control subcellular protein localization in other multicellular organisms. “The technique uses light-induced heterodimerization to control protein location and is based on the Phytochrome-PIF system of plants. I’m hoping to use it to understand the generation of neuron shape in vivo”, added co-author Rachel Moore.
Reversible optogenetic control of subcellular protein localisation in a live vertebrate embryo. Buckley CE, Moore RE, Reade A, Goldberg AR, Weiner OD, Clarke JDW (2016) Developmental Cell 36, 117-126.
This work was funded by the Biotechnology and Biological Sciences Research Council, the European Molecular Biology Organization and the Wellcome Trust.
For further information on this story or about the centre please contact Andreia Carvalho, Head of Scientific Affairs, MRC Centre for Developmental Neurobiology, King’s College London (email@example.com).
The MRC Centre for Developmental Neurobiology is part of the Institute of Psychiatry, Psychology & Neuroscience at King's College London.