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Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium

Jan N. Hansen, Fabian Kaiser, Christina Klausen, Birthe Stüven, Raymond Chong, Wolfgang Bönigk, David U. Mick, Andreas Möglich, Nathalie Jurisch-Yaksi, Florian I. Schmidt, Dagmar Wachten

Preprint posted on April 15, 2020 https://www.biorxiv.org/content/10.1101/2020.02.04.933440v2

Article now published in eLife at http://dx.doi.org/10.7554/eLife.57907

Hooked to cilia: a novel nanobody-based tool opens the way for acute manipulation of primary cilia.

Selected by Gustavo Aguilar

Background

Cells have developed an extraordinary repertoire of compartments, from cellular sub-domains to organelles, vesicles and membraneless organelles. This way, most cellular functions are restricted in space, ensuring the presence of all the required molecular players and minimising the crosstalk with other cellular processes, among other benefits. When trying to manipulate our favourite cellular activity, either to decipher its function or to exploit its possible benefits, it is critical to consider this spatial component, otherwise we could easily be mistaken in our interpretation.

A paradigmatic case of signalling compartmentalisation is the primary cilium, sometimes referred to as the “cell’s antenna”. This surface projection harbours distinct receptors and downstream signalling components in a Lilliputian reaction volume, permitting highly sensitive and fast signal transduction. Among all the cell’s second messengers, cAMP plays a central role in signal transduction in the cilium, acting downstream of diverse GPCRs. In recent years, the direct observation and manipulation of cAMP has been of major interest to understand the functioning of cilia. Currently, two light-activated enzymes permit both the production and hydrolysis of cAMP: the photo-activated adenylate cyclase (bPAC)1 and the light-activated phosphodiesterase (LAPD)2. Despite the availability of optogenetic tools, restricted manipulation of cAMP to the primary cilia has been impossible, hampering the dissection of its local functions.

 

The tool

 Hansen, Kaiser et al.3 propose a novel way to localize virtually any GFP or mCherry tagged protein to the primary cilium. They envisioned that if tagging of the protein of interest per se did not affect protein function, they could use such moiety to “drag” the protein to the cilium. To do so they made use of nanobodies, which are single-chain protein binders derived from heavy-chain only antibodies. Nanobodies have comparable binding affinity to normal antibodies, which together with its small size and solubility, makes them perfect for intracellular usage4. In their case, a nanobody recognizing GFP or mCherry was fused to a cilium-specific protein scaffold, resulting in the localization of the nanobody in the cilium. Upon co-expression of this nanobody and a GFP- or mCherry-tagged protein the latter will be re-localized to the cilium.

The authors apply this method to localize bPAC and LAPD in the cilium and this way achieve local modulation of cAMP levels.

Scheme of the new cilia-targeting approach. Extracted from Figure 2 of Hansen, Kaiser et al. 2020.

 

The specifics

The authors show that direct fusion of the bPAC and LAPD to protein domains targeted to the primary cilium (in particular the N terminal region of the mouse Nphp3) resulted in ciliary localization but reduced or impeded enzymatic activity.

To overcome this limitation, they developed a bipartite solution to target proteins of interest to the cilium. On one hand, they fused the cilia-specific “scaffold” mentioned before (truncated mNphp3) to an anti-mCherry nanobody, a highly specific protein binder that recognizes the protein mCherry with nanomolar affinity. This cilia-localised nanobody was co-expressed with the mCherry-tagged Optoenzymes bPAC or LAPD, resulting in the latter being “dragged” to the cilia by the nanobody. They confirmed that the nanobody-mediated relocalization of bPAC and LAPD resulted in fully functional enzymatic activity.

 

Relocalization of LAPD-mCherry to the cilia. Nanobody in green. LAPD-mCherry in red. Primary cilium in cyan. Dapi in blue. Modified from figure 2 of Hansen, Kaiser et al. 2020.

 

Next a FRET based reporter of cAMP was targeted to the primary cilia in a similar way. In this case, the authors used an anti-GFP nanobody instead of the anti-mCherry. mNphp3-anti-GFP nanobody fusion could bind the fluorescent proteins of the FRET reporter and localise it to the cilium. The reporter could still detect changes in cAMP concentration.

The new toolkit was then used to investigate the effect of local levels of cAMP in regulating cilia length. Their data supports that local cAMP in the cilium increase its length whereas global cAMP increase is associated with cilia shortening.

Finally, the authors showed that the nanobody-mediated cilia targeting can be used in vivo. Expression of the cilia localised anti-mCherry nanobody in zebrafish could enrich in the cilia an otherwise cytoplasmic RFP.

 

Why I think this paper is important.

 The specific subcellular targeting of functional domains is a challenge with which the authors deal elegantly. Targeting functional domains for long periods may result in unpredictable outputs. In this study, the use of optogenetic tools overcomes this problem, adding temporal control to the setup. In this and few other recent papers5,6,7 the combination of nanobodies and optogenetic tools is starting to flourish and will garner exciting methods for cell biology.

The use of nanobodies against commonly used fluorescent tags allows relocalization of many already existing GFP- and mCherry-tagged proteins to the cilia and will inspire other scientist to tackle the function of the cilia in a much more reliable and imaginative way.

 

Questions to the authors.

Recently, the list of intracellular protein binders has increased dramatically, Alphatag, Moontag and Frankenbodies are only the tip of the iceberg. These binders recognise small non-fluorescent tags. Do the authors contemplate the use of such binders to be able combine their different toolkit components at the same time? What possibilities would this open?

The validation of the cilia-targeted FRET sensor was done by stimulating the production or hydrolysis of cAMP by drugs. What do the authors think is the sensitivity of the FRET sensor? Would it be able to detect physiological changes in cAMP?

The authors also showed that localisation of bPAC to the cilia at high levels could by itself (without photo-activation) result in a change in cilia length. Could the authors elaborate on this? Does this mean that bPAC has a very low basal activity? Or could it be due to the expression of the mNphp3 scaffold?

The tools seem ready to start exploring when is cAMP produced in the cilia and dissect its role. Apart from control of cilia length, which are the biological questions where the authors think these tools will be useful in the next years?

 

References.

  1. Stierl M, Stumpf P, Udwari D, Gueta R, Hagedorn R, Losi A, Gartner W, Petereit L, Efetova M, Schwarzel M, Oertner TG, Nagel G, Hegemann P (2011) Light modulation of cellular cAMP by a small bacterial photoactivated adenylyl cyclase, bPAC, of the soil bacterium Beggiatoa. The Journal of biological chemistry 286: 1181-8
  2. Stabel R, Stüven B, Hansen JN, Körschen HG, Wachten D, Möglich A (2019) Revisiting and Redesigning Light-Activated Cyclic-Mononucleotide Phosphodiesterases. J Mol Biol 431: 3029-3045
  3. Hansen, J. N., Kaiser, F., Klausen, C., Stueven, B., Chong, R., Boenigk, W., … & Wachten, D. (2020). Nanobody-directed targeting of optogenetic tools reveals differential regulation of cilia length.BioRxiv.doi: https://doi.org/10.1101/2020.02.04.933440
  4. Helma, J.; Cardoso, M.C.; Muyldermans, S.; Leonhardt, H. (2015) Nanobodies and recombinant binders in cell biology.  Cell. Biol.209, 633–644.
  5. Deng, W., Bates, J. A., Wei, H., Bartoschek, M. D., Conradt, B., & Leonhardt, H. (2020). Tunable light and drug induced depletion of target proteins. Nature Communications11(1), 1-13.
  6. Yu, D., Lee, H., Hong, J., Jung, H., Jo, Y., Oh, B. H., … & Do Heo, W. (2019). Optogenetic activation of intracellular antibodies for direct modulation of endogenous proteins. Nature methods16(11), 1095-1100.
  7. Gil, A. A., Zhao, E. M., Wilson, M. Z., Goglia, A. G., Carrasco-Lopez, C., Avalos, J. L., & Toettcher, J. E. (2019). Optogenetic control of protein binding using light-switchable nanobodies. BioRxiv, 739201. doi: https://doi.org/10.1101/739201

Tags: camp, cilia, nanobody, optogenetics

Posted on: 5th May 2020

doi: https://doi.org/10.1242/prelights.20026

Read preprint (No Ratings Yet)




Author's response

Dagmar Wachten shared

Question 1: Recently, the list of intracellular protein binders has increased dramatically, Alphatag, Moontag and Frankenbodies are only the tip of the iceberg. These binders recognise small non-fluorescent tags. Do the authors contemplate the use of such binders to be able combine their different toolkit components at the same time? What possibilities would this open?

Answer to question 1: We would love to extend our toolkit and combine different optogenetic tools with small non-fluorescent tags. This would further improve the performance of the optogenetic tools as these tags are comparably small and do not “block” another wavelength, which is particularly important when working with light. This would also be the way to go when further applying this approach in vivo in different model organisms.

 

Questions 2: The validation of the cilia-targeted FRET sensor was done by stimulating the production or hydrolysis of cAMP by drugs. What do the authors think is the sensitivity of the FRET sensor? Would it be able to detect physiological changes in cAMP?

Answer to question 2: The mlCNBD-FRET sensor is very sensitive, detecting low nM concentrations of cAMP (Mukherjee et al., eLife 2016). In fact, that was the only cAMP biosensor we could get to work in sperm flagella to detect physiological changes in cAMP levels (Mukherjee et al., eLife 2016). The basal cAMP concentration in sperm flagella is rather low around 70-100 nM. Many other cells like HEK293 operate in the same concentration regime. In primary cilia, the basal cAMP concentration has been proposed to be in the low mM range, which would be higher than in the cell body (Moore et al., PNAS, 2018). These findings were, however, challenged by others, who did not observe higher cAMP levels in primary cilia compared to the cell body (Jiang et al., PNAS, 2019). We can tune our sensor to work efficiently in different concentration ranges and, thereby, detect physiological changes in cAMP levels in the cell body or in the primary cilium.

 

Question 3: The authors also showed that localisation of bPAC to the cilia at high levels could by itself (without photo-activation) result in a change in cilia length. Could the authors elaborate on this? Does this mean that bPAC has a very low basal activity? Or could it be due to the expression of the mNphp3 scaffold?

Answer to question 3: It has been shown before that overexpression of ciliary proteins like 5-HT6 or Arl13b causes abnormal cilia growth. Our results demonstrate that also high expression levels of only the nanobody in the cilium (fused to mNphp3(201)) increases cilia length, demonstrating that over-expression of ciliary (membrane/membrane-associated) proteins has an effect on cilia length. Thus, the amount of protein expressed in the cilium has to be carefully titrated and a thorough analysis, like we present in our manuscript, is needed to rule out any unspecific effects caused by ectopic expression of the protein and, thereby, reveal the specific contribution of a protein of interest to ciliary signaling and function.

 

Question 4: The tools seem ready to start exploring when is cAMP produced in the cilia and dissect its role. Apart from control of cilia length, which are the biological questions where the authors think these tools will be useful in the next years?

Answer to question 4: The regulation of cilia length is one important cilia function that seems to be regulated by cAMP. However, there are a number of functions like the transduction of sensory information, which seems to be associated with ciliary cAMP signaling. Our tools will allow to study these processes in the future with high spatial and temporal precision.

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