Quick Picks : Brainbow flies

Nature methods published two papers which extend brainbow-like techniques of stochastic multicolored neuronal labeling into fruit flies.  Nature’s summary explains the two methods.

 

dBrainbow expression examples

 

 

The first technique, called dBrainbow, was developed by Julie Simpson, a neuroscientist at the Howard Hughes Medical Institute’s Janelia Farm Research Campus in Ashburn, Virginia, and her colleagues². This method uses enzymes called recombinases to randomly delete some of the colour-producing genes from the string, leaving different genes next to the promoter regions in different cells. Individual cells are therefore uniquely coloured and so can be easily distinguished…

The second technique, called Flybow, was developed by Salecker and her colleagues³. They used an enzyme that ‘flips’ pairs of colour-producing genes on the string, leaving different genes next to the promoter region. The ‘flipping’ enzyme is also a recombinase, and so after being inverted, some of the colour-producing genes are randomly deleted. This ensures that all the different genes on the string can potentially end up next to the promoter, and be displayed by individual modified neurons.. Flybow uses a single string of four colours — red, green, blue and yellow.

These techniques will find use in building the structural and functional connectome of the fly.

 

Optical control of gene expression in mammalian cells

Trying to start a reboot of the posts here on Brain Windows. Lots of great stuff has come out since the last regular posting period, and unfortunately I don’t have the time to cover it all. One of the most exciting papers of the last few months was Rapid blue-light–mediated induction of protein interactions in living cells published in Nature Methods. This paper reports the  logical extension of previous technologies for photoactivatable transcription we previously covered here, here, and here.

There are two key technical improvements in the system from the Tucker Lab.  First, the genetic light switch, a cryptochrome 2 (CRY2) interaction with cryptochrome-interacting basic-helix-loop-helix protien (CIB1), is activated by blue light rather than the red light of previous switches based on phycocyanobilins.  Second, and more importantly, the cofactors necessary for the switch action (flavin and pterin chromophores), are endogenously expressed in mammalian tissues.  Thus, these switches should be usable in vivo without potentially tricky loading of the cofactors.

Upon illumination, the authors observed rapid translocation (in 1 second!) of fluorescent proteins tagged with CRY2 to cell membranes with CIB1 anchored to it.  They also were able to couple it to Gal4-UAS and Split-Cre expression systems, which let them drive reporter genes such as GFP by blue-light illumination.  I was a bit underwhelmed by the efficacy of the cre-induction, only around 15% of cells expressed the cre-driven EGFP after 24 hours of illumination, but maybe that is due to my ignorance of the current limits of the split-cre system.  That efficacy will certainly need to be improved for the REALLY cool stuff one can imagine doing with this.

What are the cool things?  Well, say you are doing some GCAMP3 imaging of a few hundred cells in the cortex during an awake behavior.  You see an ensemble of neurons whose activity is correlated to some aspect of the behavior, like a motor command, a perception or a decision. You want to prove the function of these neurons, to investigate their coding by subtracting or adding activity directly into this specific functional group. How do we control ONLY this group?

A pan-neuronal channelrhodopsin, or even one packaged in a cre-dependent virus injected into a cre reporter line will not allow you to change the spike patterns of only this ensemble. This ensemble is not differentiable from its neighbors by genetic type, only by functional relevance.  You have to hit its neighbors or shared genetic subtype with the same hammer.  But if you have one of these CRY2-CIB1 split cre switches that drive ChR2 expressed across the cortex, you could shine a blue laser (or presumably a two-photon laser) onto the members of the ensemble and turn on optical control of only that functional group.

Details of course still need to be worked out. What is the 2p cross-section of the system? How do you make it compatible with optical imaging and optical control?  How do you improve the speed and efficacy of the switch? These are things that will come with time.  The power of this technique is even recognized by apparent competitors in the field; Anselm Levskaya closed his packed SfN talk on phycocyanobilin-based optical switches with a shout out to this work.

Stay tuned…
Kennedy MJ, Hughes RM, Peteya LA, Schwartz JW, Ehlers MD, & Tucker CL (2010). Rapid blue-light-mediated induction of protein interactions in living cells. Nature methods, 7 (12), 973-5 PMID: 21037589

Light-switchable protein interactions

A fully genetically-encoded approach to light-activated transcription is getting closer now that a new, generalizable method of light-switchable protein interactions has been published.  In Nature’s advance online publication, Spatiotemporal control of cell signalling using a light-switchable protein interaction, Anselm Levskaya of the Voigt lab at UCSF and co-authors demonstrate inducible, reversible control of protein binding, localization and signalling in mammalian cells.  

apo-PhyB covalently binds to the chromophore phycocyanobilin (PCB) to form a light-sensitive holoprotein. PhyB undergoes conformational changes between the Pr and Pfr states catalysed by red and infrared light, reversibly associating with the PIF domain only in the Pfr state. This heterodimerization interaction can be used to translocate a YFP-tagged PIF domain to PhyB tagged by mCherry and localized to the plasma membrane by the C-terminal CAAX motif of Kras.

When asked about the possibility that this could be used in-vivo, Levskaya said

The only real caveat for in-vivo work is delivery of the non-native PCB tetrapyrrole. From the literature and my experience with cell culture I suspect it shouldn’t be hard to just administer it directly to animals to get saturating levels for holoprotein formation. It might even be possible just to feed animals Spirulina (where it comes from). There’s nutrition literature that suggests their livers are capable of freeing PCB and getting it into the blood stream.

 

Observing light-induced Cdc42 activation with a TIRF recruitment biosensor

Expression of genetic tools that control neural activity (Channelrhodopsins, Halorhodopsins, DREADDs) in functionally defined populations, such as neurons that are active during a particular task or thought, is the next big leap that needs to be made in systems neuroscience. This may be achieved by combining an imaging technique to identify active neurons, such as G-CaMP3, with photo-switchable transcription. The technique presented in the above paper is one promising avenue which may lead to cell-specific photo-switchable transcription.  Once robust versions of these tools are in place, scientists will begin to work out the complex and thrilling processes of reverse-engineering and manipulation of specific thoughts and memories, at least in mice and rats.

Journal Club #4 : Photoactivatable transcription

Many organisms regulate gene transcription via sunlight.  In plants, phototaxis, flowering and germination all are light dependent processes. Circadian rhythms in many species is entrained by light. Light-activated transcription is achieved through a variety of mechanisms.  Some of these mechanisms may be usable as a powerful tool to control gene expression in selected cells with high spatial and temporal resolution. When paired with other optical tools, such as genetically-encoded calcium indicators or channelrhodopsins, this technique would give unprecedented specificity in recording and manipulating brain activity. In this journal club, I review two major systems for photoactivateable transcription and their prospects for application in mammalian systems.