The function of TANGLED1
TANGLED1 (TAN1) is a microtubule binding protein with weak similarity to the microtubule binding domain of the tumor suppressor adenomapolyposis coli (APC), identified by Laurie Smith and Anne Sylvester (paper here). In vitro, TAN1 promotes angle independent microtubule bundling (paper here). 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 tan1 mutant in both maize and Arabidopsis has cells with mostly normally placed preprophase bands, but the phragmoplast is often or sometimes misoriented. How does TAN1 promote phragmoplast guidance to the division site? A deletion and localization study showed that TAN1 appears to be recruited to the division site by at least two different mechanisms because these two domains localize to the division site at distinct parts of the cell cycle (paper here). These distinct domains are differentially required to restore wild-type growth to tan1 air9 mutants in Arabidopsis, with the first ~130 amino acids of TAN1 playing an essential role (paper here). To address the function of TAN1 during telophase, we constructed a cyclin-B-destruction box fused to TAN1 to eliminate it in anaphase (the cell cycle stage before telophase), and crossed it into the tan mutant in maize. Although the cyclin-B-destruction box didn't completely eliminate TAN1, it provided 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 (paper 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 BiMolecularFluorescence 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, auxin-induced-in-roots-9 (air9) with a great collaboration with Professor Henrik Buschmann. Here is Ricardo's paper!
2) Mathematical modeling of plant cell division orientation. We developed a model that uses the 3D shape of cells to predict division plane probabilities with Professor Kenneth Brakke and many other wonderful collaborators (paper)! Additionally, we analyzed tan1 mutant divisions, and showed t
3) Identification of new division plane orientation mutants in maize and Arabidopsis thaliana.
4) We are using in vitro assays to assess how TAN1 interacts with microtubules with Professor Ram Dixit. Here is our paper showing that TAN1 bundles microtubules in vitro.
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.