Category Archives: Neuroscience

Local translation of ribosomal proteins in axons

A recent bioRxiv pre-print publication from Christine Holt’s lab suggests that ribosomes may be remodeled in axons by locally translated ribosomal proteins. This is surprising because we know that ribosomes are assembled in the nucleolus. Well, I have some concerns about a few of the experiments depicted there.

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This month’s Nature methods (part 2): optogenetics

Optogenetic tools are light-sensitive genetically encoded proteins that, upon light activation, affect a molecular change in the cells. In the previous post I described an optogenetic system to induce transcription. However, the most common use is of channelrhodopsin (ChR) molecules, that alter ion homeostasis upon illumination, and when expressed in neurons, can affect neuronal activity and – in live animals – a change in behavior.

Though optogenetics were used extensively in neurons in culture or in mice, its use was limited in flies. The reason was that the most common ChRs are activated by blue light, which does not penetrate well into the brains of live flies. Therefore, theremogenetic control has been used. In thermogenetics, a thermosensitive channel is expressed in the neurons. However, theremogenetics is far less accurate than optogenetics in terms of temporal resolution and intensity. On top of that, one needs to consider the effect of changes in heat on behavior.

To overcome that, the group of David Anderson decided to use a newly developed red-shifter ChR, ReaChr.

Using ReaChr, but not other Chrs, the researchers were able to activate specific neurons and alter fly behavior. Here’s one of their videos. Using this tool, they learned about the sexual behavior of the male fly and came to the conclusion that there are two sets of neurons which can be separated, with social experience affecting only one set of neurons.

The second optogenetic paper in this issue is more technical. The group of Edward Boyden decided to screen for new candidate ChRs in over 100 species of alga. They sequenced transcriptomes of 127 alga species and isolated 61 candidate ChRs which they tested for light-induced currents using electrophysiology methods. They chose 20 candidates and tested them in a range of wavelength excitations looking at current maxima, kinetics and more parameters.

characteristics of selected channelrhodopsins from different alga species. A, B, C, D - currents amplitude at different excitation wavelengths in HEK293 (A-C) and neurons (D).  E - Chrimson is the most red-shifted ChR. F - off kinetics. G - on-kinetics. H - recovery kinetics.

characteristics of selected channelrhodopsins from different alga species. A, B, C, D – currents amplitude at different excitation wavelengths in HEK293 (A-C) and neurons (D). E – Chrimson is the most red-shifted ChR. F – off kinetics. G – on-kinetics. H – recovery kinetics. Source: Klapoetke et. al. (2014) Nat. Meth. 11:338-346.

Of these, they selected two unique ChR: Chronos (with very high activity after blue or green light excitation) and Chrimson (the most red-shifted ChR – 45nm more red-shifted than ReaChR). After further characterization they showed that Chrimson is a good optogenetic tool for live flies and showed that they can stimulate neurons in fly brains.

Last, they created a two-color system. One of the limitations of the current ChRs is that all of them can be stimulated to some extent by blue light. Although Chrimson can be activate by blue light, the kinetics of Chronos are 10 fold faster. They did a very detailed work in finding the best conditions for a two-color system, based on the excitation pulse and power as well as expression level of the different ChRs.

All in all, a very nice work.

 

 
ResearchBlogging.orgKlapoetke NC, Murata Y, Kim SS, Pulver SR, Birdsey-Benson A, Cho YK, Morimoto TK, Chuong AS, Carpenter EJ, Tian Z, Wang J, Xie Y, Yan Z, Zhang Y, Chow BY, Surek B, Melkonian M, Jayaraman V, Constantine-Paton M, Wong GK, & Boyden ES (2014). Independent optical excitation of distinct neural populations. Nature methods, 11 (3), 338-46 PMID: 24509633
Inagaki HK, Jung Y, Hoopfer ED, Wong AM, Mishra N, Lin JY, Tsien RY, & Anderson DJ (2014). Optogenetic control of Drosophila using a red-shifted channelrhodopsin reveals experience-dependent influences on courtship. Nature methods, 11 (3), 325-32 PMID: 24363022
 

 

Looking at single mRNAs in neurons hints at memory formation

It is postulated that learning and memory are modulated by synaptic plasticity – molecular changes  that result in changes in the synapse morphology and signaling capacity. Local protein translation is considered important for synaptic plasticity. Two works from our lab were published last month (back to back!) in Science. Both papers deal with how beta-actin mRNA localization and dynamics in neurons may account for local protein translation upon stimulation, and hence, may supply insight into memory formation.

The first paper by Adina Buxbaum shows that beta-actin mRNAs in dendrites are “unmasked” upon activation of the dendrites. Using single molecule FISH, She noticed that the average number of probes bound to the mRNAs in dendrites (but not in adjacent glia cells) was lower than expects, and this number increased upon stimulation. Not only that, there were more mRNAs in the stimulated dendrites. This indicated masking by a protein “coating” that prevented FISH probe binding in the unstimulated cells. A modified FISH protocol which included a protease digestion step prior to probe hybridization showed that indeed the mRNAs were masked by proteins.

single molecule FISH for beta-actin mRNA in dendrites shows that mRNAs in unstimulated neurons are masked. A) Unstimulated neuron. B) stimulated neuron showing increased number of spots. C) Unstimulated neuron, in which the fixed cells were digested with protease prior to FISH probe hybridization. Source: Buxbaum, Wu & Singer (2014). Science Vol. 343  pp. 419-422

single molecule FISH for beta-actin mRNA in dendrites shows that mRNAs in unstimulated neurons are masked. A) Unstimulated neuron. B) stimulated neuron showing increased number of spots. C) Unstimulated neuron, in which the fixed cells were digested with protease prior to FISH probe hybridization. Source: Buxbaum, Wu & Singer (2014). Science Vol. 343 pp. 419-422

 

She further showed that this masking relates to other mRNAs, as well as to ribosomes, and that this is due to a metabolic process resulting from stimulation. Thus, this unmasking process may be a way to “activate” localized mRNAs for translation.

Apart from being a very neat paper technically and biologically, I think it was exceptionally entertaining to begin her paper by quoting an 1894 work by Cajal, the father of neuroscience.

The second paper by Hye-Yoon Park follows the dynamics of single molecule endogenous beta-actin mRNAs in neurons by live imaging, using the MS2 system. She shows movement of mRNAs along dendrites, as well as some events of merging or splitting – suggesting that some mRNAs are packed together in larger granules – which may regulate local translation. She also looked at brain slices, visualizing beta actin transcription dynamics. This is an important achievement since it is much harder to look at mRNA dynamics in tissue slices than in single cells on plate, due to background fluorescence. Though some biological insight is derived here, this is more of a “new technology” report.

Live imaging of beta-actin mRNAs in dendrites (movie. Source: Park HY et al. (2014) Science Vol. 343 pp. 422-424)

These papers are just the beginning of a long-term story of how mRNA localization and local translation are regulated in neurons.  A lot of cool experiments are being done in our lab in this regard and I’ll report more as they are published.

ResearchBlogging.orgBuxbaum AR, Wu B, & Singer RH (2014). Single β-actin mRNA detection in neurons reveals a mechanism for regulating its translatability. Science (New York, N.Y.), 343 (6169), 419-22 PMID: 24458642
Park HY, Lim H, Yoon YJ, Follenzi A, Nwokafor C, Lopez-Jones M, Meng X, & Singer RH (2014). Visualization of dynamics of single endogenous mRNA labeled in live mouse. Science (New York, N.Y.), 343 (6169), 422-4 PMID: 24458643

Update

Hello dear readers,

As you’ve probably noticed, I’ve been away for a couple of months. Just overload of work and other stuff that came in the way of updating. I hope to return to my regular reports on interesting papers (which have accumulated on my desk).

The meeting last month on trafficking was very interesting, though not as much microscopy talks as I thought. Nothing really ground breaking in terms of imaging methods.

Orna Amster Choder showed beautiful images of mRNA localization in bacteria, and Eitan Bibi talked about the targeting of mRNA and ribosomes to the E. coli plasma membrane.

Yaron Shav-Tal used imaging to look at transcription dynamics of Cyclin D1, as well as upstream events (i.e. the signal transduction process leading to transcription activation).

Jeff Gerst & Yoav Arava talked about localization of mRNAs and local translation of mRNAs to mitochondria* in yeast.

* Actually, the mito people at the meeting said that nowadays it should be called “mitochondrion” (a single mitochondria) since this is just one organelle spread throughout the cell.

Hagai Abeliovich talked about mitophagy, which is apparently dependent on mitochondrial dynamics and relates to hypo and hyper-polarization of mitochondria “daughters” during fission events.

Importantly, I learnt from him that GFP is stable in the yeast vacuole (the equivalent of lysosome) which is very interesting and maybe useful info in yeast imaging.

Felippe court and Jose Sotelo talked about movement of RNA from Glia cells to Axons. Very exciting stuff, but still missing a stronger evidence for the movement, and particularly biological effect (translation…).

I have learnt that the mito makes contacts with the vacuole and the ER membranes, where they may exchange lipids and proteins.

And there were many more interesting talks. It was a good meeting.

The next meeting I’m going to attend is FISEB 2014 where I will also present my work in an oral presentation (its called “oral poster”).

Looking through the brain with CLARITY!

Imaging single layers of cells is very easy since the light can penetrate the cells quite easily. Imaging a tissue sample of several layers of cells is more difficult, because the passage of light is gradually blocked. To image a whole organ without slicing it was near-impossible, until now.

CLARITY is a new method, developed by the lab of Karl Deisseroth, a neuroscientists from Stanford university and published in the Nature Online. In essence, they took a whole mouse brain, infused it with a hydrogel, which was cross-linked to all the “important” biomolecules (proteins & nucleic acids). They then removed all the lipids and other “non-important” molecules (which were not cross-linked to the hydrogel), with a detergent, SDS.

The result was a transparent mouse brain, that maintained all of it original cellular (and presumably sub-cellular) structure.

Mouse brain before & after CLARITY. Source: Chung, K. et al Nature (2013)

They could then image into the brain, using pre-expressed fluorescent proteins, immunofluorescence (IF) or FISH. Importantly, they claim that one can perform several rounds of IF (and similarly FISH) and they show images to proved that.

They also show that the signal intensity of the IF signal does not diminish when going deeper into the tissue, or using different z-sections.

Structural and molecular phenotyping of a piece of human brain using CLARITY. Source: Chung, K. et al Nature (2013).

Structural and molecular phenotyping of a piece of human brain using CLARITY. Source: Chung, K. et al Nature (2013).

It is my understanding that people in the Neuroscience field have known about CLARITY for quite some time, just waited for it to get published already. It IS very exciting and amazing.

Watch the Clarity Video:

Once CLARITY becomes widespread, we should get immense amount of data on the cellular & molecular structure of many types of whole (unsliced) tissues.  We could follow development, disease and much more.

Perhaps one day even whole animals will be imaged.

ResearchBlogging.orgChung, K., Wallace, J., Kim, S., Kalyanasundaram, S., Andalman, A., Davidson, T., Mirzabekov, J., Zalocusky, K., Mattis, J., Denisin, A., Pak, S., Bernstein, H., Ramakrishnan, C., Grosenick, L., Gradinaru, V., & Deisseroth, K. (2013). Structural and molecular interrogation of intact biological systems Nature DOI: 10.1038/nature12107

Watching Neurons in action

Fluorescent sensors are important tools that can allow real-time, live, single molecule imaging of microscopic millisecond scale events. It is even better if these sensors are genetically encoded sensors (i.e. fluorescent proteins). We have already encountered the pH sensors pHluorin and pHTomato and the Ca2+ sensor GCaMP. There have been a few others, such as HyPer that detects H2O2 or ArcLight and ElectrikPk which are voltage sensors.

Now, the group of Loren Looger from HHMI Janelia  developed a sensor for a very important molecule: L-glutamate. Continue reading

Optogenetics blog

 Open optogenetics blog, is a blog about optogenetics. Optogenetics is a rather new field in neurobiology and fluorescent microscopy. The main idea of optogenetics is to have genetically encoded optically controled proteins (mostly, ion channels) and fluorescent sensors. I briefly discussed it here.
The blog discusses new sensors and other optogenetic research and papers. Recommended!