The function of TANGLED
TANGLED (TAN) is a microtubule binding protein with weak similarity to the microtubule binding domain of the tumor suppressor adenomapolyposis coli (APC). It plays a role in promoting phragmoplast guidance to the division site. Here is a paper describing the molecular identification of TANGLED1 in maize. The tan mutant in both maize and Arabidopsis has cells with normal preprophase bands, but the phragmoplast is often misoriented. How does TAN promote phragmoplast guidance to the division site? Intriguingly, from a deletion and localization study (paper here), we showed that TAN appears to be recruited to the division site by at least two different mechanisms because two domains (fused to the yellow fluorescent protein, YFP) localize to the division site at temporally distinct parts of the cell cycle. To address the function of TAN during telophase, we constructed a cyclin-B-destruction box fused to TAN to eliminate it in anaphase (the cell cycle stage before telophase), and have crossed it into the tan mutant. Although the cyclin-B-destruction box didn't work as we anticipated, we discovered a way to separate cell cycle defects from division plane defects. A combination of division plane defects and cell cycle delays causes the short stature of the tan1 mutant in maize. Check out the paper on TAN1 function in maize here!
The Division site interactome
Several proteins have been identified as putative TANGLED interacting proteins by immunoprecipitation followed by mass spectrometry (in collaboration with Professor Steve Briggs at UCSD) or by yeast-two hybrid. Interestingly, many of the proteins identified interact with a specific domain of TANGLED which we demonstrated localizes to the division site during telophase. Our goal is to confirm these interactions using co-immunoprecipitation and Bi Molecular Fluorescence Complementation (BiFC , otherwise known as "Split YFP"). Using a reverse genetics pipeline, the phenotype of the mutants in putative interacting partners are assessed for defects in division plane orientation.
Thanks to a tremendous collaboration with Professor Bing Yang, we used CRISPR/Cas9 (review here) to generate mutants of some of the TAN1 interactors in maize.
1) Assessing an enhancer of the tan1 mutant in Arabidopsis thaliana (and using it as a tool to understand TAN1 function in vivo). Great collaboration with Professor Henrik Buschmann. Here is Ricardo's paper!
2) Mathematical modeling of plant cell division orientation. Here is the preprint.
3) Identification of new division plane orientation mutants in maize.
This is a timelapse movie of a tangled mutant cell expressing a live cell marker for microtubules. This cell starts in prophase, and then progresses until cytokinesis is completed. Note that the phragmoplast does not reach the correct division site.