Who: I am a life-sciences explorer.
What: I surf nutrient-to-gene trails.
Where: My base camp is at the University of Geneva.
When: - gianpaolo.rando@unige.ch,

Photo-activable reporters in space and time

Photoactivation is the property of a molecule of being capable of pronounced changes in its chemical properties in response to irradiation with light of a specific wavelength and intensity. This feature provides unique possibilities to decipher molecular pathways with enhanced temporal and spatial resolution.

Proteasome temporal dynamics
Fluorescent reporters have already been used in the past to monitor proteosomal degradation, but effects on reporter degradation are difficult to separate from effects on reporter synthesis. Hamer and colleagues designed a reporter in which these processes can be easily distinguished by changing the color of already-translated proteins. The green-to-red photoconvertible protein Dendra2 is fused to a form of ubiquitin that targets Dendra2 for proteosomal degradation. By observing only the photoconverted red form of the reporter, the effects on the reporter synthesis can be ruled out. The researchers used this reporter to monitor the cell type- and age-dependence of ubiquitin proteosome activity in living C.elegans. (Hamer et al., Nature Methods 2010). In this case, photo-activation changes the color of Dendra2.

Lymph-node spatial dynamics
Germinal centers of lymph nodes are a site of antibody affinity maturation: in these sites, lymphocyte development goes through a selection of proliferating B cells through both clonal expansion and affinity maturation. At the end of this process, B cells carrying high-affinity antibodies are selected. Unfortunately, we don't know so much about the dynamics of this selection, so  we are still missing the full comprehension of a process that is fundamental to our immunity. In a new Cell paper,  Gabriel Victoria and colleagues describes the generation of a new reporter mouse made to follow with great space-temporal resolution the maturation of lymphocytes in the germinal center. This transgenic mouse expresses photo-activable PA-GFP in all hematopoietic cells (via ubiquitin C promoter). With this reporter mouse, it is now possible to photo-activate GFP-lymphocytes with a great micro-anatomical precision of ~10 um (see picture).

Spatial precision (X-Y axis) multiphoton photoactivation of inguinal lymph nodes from PA-GFP transgenic mice imaged at λ = 940 nm before and after photoactivation at λ = 830 nm of a "GFP" region of interest.
Then, one can track lymphocyte movements into the germinal centers over many hours. By doing so, one can draw a map of cellular migration in the germinal center. Based on this map, lymphocites were taken at critical time points, FACS separated, and gene expression finally analyzed by microarray analysis. The final super-imposition of gene ontologies for cellular proliferation on the spatio-temporal cartography of cellular movements in the lymphocyte shines a light on B cell maturation (Victora et al., Cell 2010). In this case, photoactivation make fluorescent a protein that was not fluorescent before. 

The smaller photoactivable reporter is a nucleoside long!
UV-light irradiation has been used in the past to isolate RNAs bound to specific binding proteins (RBPs) but the discovery rate was inefficient because of problems in separating signal from background. Haffner and colleagues used photoactivable nucleosides as efficient and non-toxic crosslinkers that are well incorporated into the nascent RNA. Cells are grown in the presence of X mM of 4-thiouridine (4SU): 4SU-substituted RNA is 'activated' by UV light at 365 nm and specifically crosslinks to its RNA-binding partner proteins. Interestingly, if crosslinked and de-crosslinked (after immunoprecipitation of the RNA-bound-protein) 4SU is then mis-paired with guanine (G) instead of adenine (A) during the reverse transcription to cDNA. This allows the precise mapping of the binding sites by scoring the G/A transitions in the cDNA sequence. The researchers used this technique to define the binding motifs of 13 RNA-binding proteins. (Haffner et al., Cell 2010) In this case, photoactivation changes the chemical reactivity of small molecules making a protein-trap.

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Hafner et al. (2010). Transcriptome-wide Identification of RNA-Binding Protein and MicroRNA Target Sites by PAR-CLIP Cell, 141 (1), 129-141 DOI: 10.1016/j.cell.2010.03.009

Hamer, et al. (2010). A photoconvertible reporter of the ubiquitin-proteasome system in vivo Nature Methods, 7 (6), 473-478 DOI: 10.1038/nmeth.1460

Victora et al. (2010). Germinal center dynamics revealed by multiphoton microscopy with a photoactivable fluorescent reporter. Cell, 143(4), 592-605. DOI: 10.1016/j.cell.2010.10.032