An In Vivo Chitin Visualization Tool

Chitin

Sobala et al (2015) published the results of their efforts to develop a system that enables them to visualize the location of chitin in living insects. While they developed and tested their tool in Drosophila melanogaster it is designed in such a way as to likely make it useful in any insect that can be transformed.

The authors review the various approaches and strategies that exist for localizing chitin in cells and tissues. Major limitations include lack of specificity in some cases and the need to work with fixed tissues.

To overcome existing technical limitations the authors developed a genetic in vivo chitin reporter system.

The design strategy was conceptually straightforward – express and secrete a fusion protein consisting of a strong fluorescent protein reporter and a protein domain that has chitin binding activity.

Section of insect integument By Xvazquez (File:Cuticula.svg on wikipedia) [CC BY-SA 3.0 (http://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons

To execute this design the authors constructed a reporter protein composed of the fluorescent protein td-Tomato (which has a high fluorescence output) and two additional protein domains. The first consisted of a segment of the Dy1 protein that directs the protein to be secreted apically. The second protein domain consisted of a chitin binding domain from the chitinase A1 protein found in Bacillus circulans.

Through a series of well-controlled experiments the authors were able to show that their fusion protein – referred to as ChtVis-Tomato – was secreted apically and that it binds to chitin.  As a control the authors constructed an identical reporter but lacking the chitin binding domain.

ChtVis-Tomato from Sobala et al (2015)

Chitin binding appeared to be specific and the authors presented some data that suggested that some of ChtVis-Tomato was stably bound to chitin while some seemed to be continually binding and releasing from chitin.

ChtVis-Tomato was useful for making long time lapse observations in vivo.

The ChtVis-Tomato reporter was construted in the pWALIUM10-moe vector and inserted into the Drosophila genome using phiC31 mediated integration. ChtVis-Tomato is under the regulatory control of a 5XUAS-containing promoter allowing it to be expressed in any Gal4 driver lines of D. melanogaster. The reporter is flanked by gypsy insulator sequences. pWALIUM10-moe contains a mini white gene that serves as a genetic marker in the appropriate mutant white background.

With a bit of modification and reconfiguring ChtVis-Tomato could easily be deployed in any insect and is expected to function as it has in D. melanogaster.

Lukasz F. Sobala, Ying Wang, Paul N. Adler (2015) ChtVis-Tomato, a genetic reporter for in vivo visualization of chitin deposition in Drosophila. Development 2015 142: 3974-3981; doi: 10.1242/dev.126987

 

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