This is my personal web page describing some of the optogenetics projects that I have worked on in the past years.
Cellular (subcellular) Optogenetics
Our work focusses on the development and application of cellular optogenetics (different from classical optogenetics, that uses light controlled ionchannels to control the activity of neurons). We utitilize plant light receptor domains to build light controllable versions of proteins that we are interested in. Such light controllable proteins can be turned off or on in split second, and allow us to control proteins at the subcellular level. One of the light receptor domains that we utilize is called a LOV2 domain. This domain is part of a plant protein named phototropin, which in plants is essential for growth towards sunlight (phototropism). The LOV2 domain is particularly sensitive to blue light.
Here's a link to our recent review: "Lights, cytoskeleton, action: Optogenetic control of cell dynamics"
In this review we discuss various strategies that have been employed to optogenetically control cytoskeleton/ cell dynamics.
Current Opinion in Cell Biology (click here)
fig 1 preview: comparison of various light receptors.
A) A.s LOV2(phot1) structure in the dark(top) and lit(bottom) states B) A.t Cry1-PHR C) A.t PhyB-PSM D) structure of GFP for comparison.
Below is a link to our paper on a blue light inactivated version of the microtubule plus-end-tracking protein EB1, which was published in Nature Cell Biology in 2018. EB1 is an important regulator of the microtubule cytoskeleton in eukaryotes. It can regulate MT growth dynamics and it can mediate attachement of MTs to other subcellular structures, by recruiting over 40 different types of +TIPs to growing MT ends. We engineered a variant of EB1 which splits apart in the presence of blue light, and can no longer recruit these other +TIPs to the ends of MTs. This impacts MT growth, and MT attachment at the cell cortex. We engineered pi-EB1 by inserting a light sensitive protein module in the EB1 protein. This module consists of the plant light receptor domain LOV2, plus a a small protein, named Zdk1, that binds LOV2 in the dark but dissociates in the presence of blue light (LOVTRAP). Below you find a schematic of photo-inactivated(pi)-EB1, and images of a cell before and after inactivation of pi-EB1.
van Haren J, Charafeddine RA, Ettinger A, Wang H, Hahn KM, Wittmann T. Local control of intracellular microtubule dynamics by EB1 photodissociation. Nature Cell Biology, 2018 Mar 20; (3):252-261. doi: 10.1038/s41556-017-0028-5. Epub 2018 Jan 29.
Nature Cell Biology, Article (click here)
We also published supporting protocols on how to make cell lines expressing this optogenetic switch, and provide some advice on things to keep in mind when designing your own optoswitches using this "domain splitting" strategy.
Wittmann T, van Haren J. Generation of cell lines with light-controlled microtubule dynamics. Protocol Exchange, 2018, published online 29 January 2018. doi:10.1038/protex.2017.155
Protocol Exchange, Protocols (click here)
pi-EB1 constructs are available on Addgene (click here)
Practical implementation of optogenetics and live cell microscopy
We recently published a chapter in
In the near future i'll try to upload more detailed step-by-step instructions on how to assemble the LEDcube
For more info also see:
- Our work was highlighted on: the Addgene blog.
- Mightex application note:"Spatial control of microtubule dynamics at the subcellular scale by patterned illumination"