Biological Projects

The microtubule-associated protein CC1

The microtubule-associated protein Companion Of Cellulose Synthase 1 (CC1) is a transmembrane protein that controls microtubule bundling and dynamics to sustain plant growth under salt stress. In collaboration with plant biologists at the University of Melbourne, our group first described the structural properties and molecular mechanisms of the interactions of the cytoplasmic domain of CC1 and microtubuli in A. thaliana (Kesten et al. 2019). Remarkably, CC1 shares many of these features with preeminent human Tau, another microtubule binding protein well characterized in the context of neurodegenerative disease, an indication of convergent evolution.

After the initial characterization, our focus is now on the regulation of CC1 activity. Intrinsically disordered domains like the N-terminal region of CC1 are often subject to post-translational modification that modify their function. The plant kinase BIN2 is a promiscuous enzyme analogous to human GSK3. Initial assays by Mass Spectrometry and mutagenesis indicate up to 11 phosphorylation sites. These are often too close in sequence to be resolved, but most importantly MS does not provide quantitative information neither on the kinetics, nor the thermodynamics (e.g. the substrate distribution).

Using real-time solution 2D and 3D NMR, we have monitored the phosphorylation reaction of CC1 for 24 – 48 hours. We are able to identify 5 main phosphorylation sites, all clustered in the N-terminus near microtubule binding motifs, and an Arginine shifting oddly (Fig. 1). The evolution of the intensities of the new peaks shows three regimes: fast phosphorylation kinetics for S32, slow for S20, and intermediate for the rest, including the perturbation of R33 (Fig. 2). The decaying counterparts confirm the results.
Mapping the data back to the sequence suggests that two N-terminal microtubule binding domains can be regulated by phosphorylation, one of them potentially subject to both fast and slow cues. Confronting with Tau, the evolutionary confluence is further supported by these mechanisms.


Kesten C, Wallmann A, Schneider R, McFarlane HE, Diehl A, Khan GA, van Rossum BJ, Lampugnani ER, Szymanski WG, Cremer N, Schmieder P, Ford KL, Seiter F, Heazlewood JL, Sanchez-Rodriguez C, Oschkinat H, Persson S (2019) The companion of cellulose synthase 1 confers salt tolerance through a Tau-like mechanism in plants. Nat Commun 10(1), 857. DOI: 10.1038/s41467-019-08780-3


Fig 1: 2D 1H-15N spectra of CC1 at t=0 and t=24 hours of incubation with BIN2 kinase inside the NMR spectrometer.

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Fig. 2: Experimental and fitted peak intensities of selected peaks over the course of experiment shown in Fig. 1

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Fig 3: Cartoon representation of the observed phosphorylation dynamics. In green, phosphorylated residues in CC1 and validated phosphorylation sites in Tau. In red, residues whose signals that decay significantly upon phosphorylation.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
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