Spatiotemporal Signalling Control
With a protein pair
Much has been learned about cells and their proteins using optical reporters such as GFP,
but scientists want to know more. Work is now aimed at using light not only to observe
cellular functions but also to control them in very precise ways. Optical manipulation of
cell function is being exploited in cutting edge research to control behavior in organisms,
characterize signaling pathways and test cellular network models. The approach has been
dubbed "Optogenetics" and was named "Method of the Year 2010" by Nature publishing.
Researchers from the University of California, San Francisco, used
the phytochrome signaling network of plants to develop a genetically
encoded light-controlled system for fine spatial and temporal control
over proteins. The Mosaic Photostimulation System was a key part
of the optical setup, allowing them to achieve tight spatial patterns of
protein recruitment in mammalian cells.
Phytochromes are photoreceptive signaling proteins that control
many light-sensitive processes in plants by detecting red and near
infrared light. The researchers optimized phytochrome B (PhyB)
and phytochrome interaction factor 3 (PIF3), to produce a photosensitive
pair, Phy-PIF, which binds in response to red light (650 nm)
and dissociates in response to infrared light (>750 nm). When PhyB
is membrane-bound, then fluorescently labelled PIF3 can be seen
to translocate to the plasma membrane under 650 nm illumination,
where the Phy-PIF complex forms.
They tested the light-switchable protein pair by locally inducing
recruitment of fluorescently-tagged Phy-PIF with a MicroPoint
microscope laser system, which allowed laser illumination with near
diffraction limited geometries and viewing of the laser irradiation in
real time. The researchers centered continuous 20 Hz pulses of 650
nm light from a UV-pumped Rhodamine 650 nm dye cell laser at
low intensity on an area of plasma membrane, while the whole cell
was simultaneously exposed to inhibitory IR light from a filtered
brightfield source at maximal intensity. TIRF imaging allowed them
to tell if the recruited fluorophore was in the membrane rather than in
the cytoplasm because it images thin cellular regions.
The researchers then developed a fully automated method to expose the
cell to both wavelengths of light. "The main challenge for developing
the optical setup was simultaneous inverse patterns of red and infrared
light," said Dr. Orion Weiner, a member of the research team. "To
combat lateral diffusion of plasma-membrane bound PIF-YFP, we
needed a zone of recruitment (red light) surrounded by an inverse
zone of infrared light." To accomplish this researchers worked with
Photonic Instruments to develop a "Complementary Mosaic Photostimulation System" that coupled the red light to the "on" state of
the device's mirrors and coupled a separate infrared light source to the
"off" state of the mirrors. In this configuration the device acts to both
"target" activity in user-defined regions and "silence" activity outside
of those regions.
The Mosaic Photostimulation System contains an array of hundreds
of thousands of microscopic semiconductor-based mirrors known as
a Digital Micromirror Device. The hinge-mounted mirrors can be individually tilted very quickly and efficiently allowing generation of
red pixels and an inverse pattern of infrared pixels at the same time.
"There is no other commercial device that can generate simultaneous
inverse patterns of red and infrared light," said Weiner. "This was
essential for our ability to generate tight spatial patterns of protein
recruitment in mammalian cells with the Phy/PIF system."
Using the Mosaic Duet they could even project a simple pixel-based
movie onto the cell membrane. TIRF imaging of the cell membrane
using an Andor iXon EMCCD camera showed that the illumination
pattern produced features as small as 3 μm. The iXon camera allowed
them to use as little fluorescence excitation light as possible, which
was desirable because the imaging wavelengths also slightly activate
the photoactivatable system said Anselm Levskaya, who led the
research team .
The researchers could also use software to "dither" the average
amount of red light in the target mask, allowing them to smoothly
titrate the fraction of active Phy and recruited PIF-YFP. This showed
that the technique could be used for 'grayscale' control of the chemical
The genetically encoded, light-switchable Phy- PIF interaction module
the researchers developed had a titrated and reversible interaction and
could potentially be used to control any live cell process that depends
on a recruitment event. Because the light can be controlled with high
spatial and temporal resolution highly complex spatial or temporal
patterns can drive a process using the Phy-PIF module.
The researchers are currently using the system with closed-loop
control to automatically tune activation of the photo-modulated
signal transduction pathway. This is important for implementing
quantitative microscopy experiments with single cells, said Levskaya.
They foresee the Phy-PIF module being useful for controlling a wide
variety of cell biological processes without requiring case-by-case
"I think one of the biggest applications it will have will be in perturbing
gene transcription and signal transduction pathways in developing
animals," said Levskaya. "Being able to alter in time and space what
signals and genes are being made should allow us to perform very
novel kinds of experiments during development to try to reverse
engineer the machinery that establishes the animal body plan."
- Nikon TiE inverted microscope with multi-line laser module and motorized TIRF illuminator
- Metamorph imaging software with EMCCD camera
- Mosaic Duet DMD AI device with Sutter DG4 light source enabling 650 nm excitaory illumination and
750 nm silencing illumination