The fundamental process of polarised exocytosis requires the interconnected activity of molecular motors trafficking vesicular cargo within a dynamic cytoskeletal network. In plants, few mechanistic details are known about how molecular motors, such as myosin XI, associate with their secretory cargo to support the ubiquitous processes of polarised growth and cell division. Live-cell imaging coupled with targeted gene knockouts and a high-throughput RNAi assay enabled the first characterisation of the loss of Rab-E function. Yeast two-hybrid and subsequent in silico structural prediction uncovered a specific interaction between Rab-E and myosin XI that is conserved between P. patens and A. thaliana. Rab-E co-localises with myosin XI at sites of active exocytosis, and at the growing tip both proteins are spatiotemporally coupled. Rab-E is required for normal plant growth in P. patens and the rab-E and myosin XI phenotypes are rescued by A. thaliana's Rab-E1c and myosin XI-K/E, respectively. Both PpMyoXI and AtMyoXI-K interact with PpRabE14, and the interaction is specifically mediated by PpMyoXI residue V1422. This interaction is required for polarised growth. Our results suggest that the interaction of Rab-E and myosin XI is a conserved feature of polarised growth in plants.
Our ability to identify genes that participate in cell growth and division is limited because their loss often leads to lethality. A solution to this is to isolate conditional mutants where the phenotype is visible under restrictive conditions. Here, we capitalize on the haploid growth-phase of the moss Physcomitrella patens to identify conditional loss-of-growth (CLoG) mutants with impaired growth at high temperature. We used whole-genome sequencing of pooled segregants to pinpoint the lesion of one of these mutants (clog1) and validated the identified mutation by rescuing the conditional phenotype by homologous recombination. We found that CLoG1 is a novel and ancient gene conserved in plants. At the restrictive temperature, clog1 plants have smaller cells but can complete cell division, indicating an important role of CLoG1 in cell growth, but not an essential role in cell division. Fluorescent protein fusions of CLoG1 indicate it is localized to microtubules with a bias towards depolymerizing microtubule ends. Silencing CLoG1 decreases microtubule dynamics, suggesting that CLoG1 plays a critical role in regulating microtubule dynamics. By discovering a novel gene critical for plant growth, our work demonstrates that P. patens is an excellent genetic system to study genes with a fundamental role in plant cell growth.
The fundamental eukaryotic process of intracellular trafficking requires the interconnected activity of molecular motors trafficking vesicular cargo within a dynamic cytoskeletal network. However, in plants, few mechanistic details are known about how molecular motors associate with their secretory cargo to support the ubiquitous processes of polarized growth and cell division. A yeast two-hybrid screen of a Physcomitrella patens library identified a RabE GTPase as an interactor of myosin XI and subsequently demonstrated all five RabE members interact with myosin XI. Consistent with a role in polarized transport, we observed RabE at the growing cell apex and at the expanding cell plate during cell division. An in vivo cross-correlation analysis of fluorescently tagged RabE and myosin XI revealed that both species are spatiotemporally coupled, demonstrating their simultaneous involvement in polarized growth. To determine if myosin XI and RabE are directly coupled, we first computationally predicted myosin XI:RabE interface through a homology modeling-directed approach. We identified a structurally conserved residue on myosin XI, V1422, that when mutated abolished RabE binding in the yeast two-hybrid system and resulted in unpolarized plants instead of the characteristic network of filamentous cells when regenerated from single cells. Together, this work demonstrates the requirement of a direct myosin XI:RabE interaction for polarized growth in plants. Fendrych, M., Synek, L., Pečenková, T., Toupalová, H., Cole, R., Drdová, E., Nebesářová, J., Šedinová, M., Hála, M., and Fowler, J.E. (2010). The Arabidopsis exocyst complex is involved in cytokinesis and cell plate maturation. Plant Cell 22, 3053-3065. Furt, F., Lemoi, K., Tuzel, E., and Vidali, L. (2012). Quantitative analysis of organelle distribution and dynamics in Physcomitrella patens protonemal cells. . PHYML Online--a web server for fast maximum likelihood-based phylogenetic inference. Nucleic Acids Res. 33, W557-559. Guindon, S., Dufayard, J.F., Lefort, V., Anisimova, M., Hordijk, W., and Gascuel, O. (2010). New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59, 307-321. Hammer, J.A., and Sellers, J.R. (2012). Walking to work: roles for class V myosins as cargo transporters. Nat Rev Mol Cell Bio 13, 13-26. Hashimoto, K., Igarashi, H., Mano, S., Takenaka, C., Shiina, T., Yamaguchi, M., Demura, T., Nishimura, M., Shimmen, T., and Yokota, E. (2008). An isoform of Arabidopsis myosin XI interacts with small GTPases in its C-terminal tail region. J. Exp. Bot. 59, 3523-3531. Heider, M.R., and Munson, M. (2012). Exorcising the exocyst complex. Traffic 13, 898-907. Hepler, P.K., and Winship, L.J. (2015). The pollen tube clear zone: Clues to the mechanism of polarized growth. . (2018). The Physcomitrella patens chromosome-scale assembly reveals moss genome structure and evolution. Plant J 93, 515-533. Lepore, D., Spassibojko, O., Pinto, G., and Collins, R.N. (2016). Cell cycle-dependent phosphory...
Individual neuron or muscle cells express many G protein coupled receptors (GPCRs) for neurotransmitters and neuropeptides. It remains unclear how these cells integrate multiple GPCR signals that all must act through the same few G proteins. We investigated how two serotonin GPCRs, Gαq-coupled SER-1 and Gαs-coupled SER-7, function together on the C. elegans egg-laying muscles to promote contraction and thus cause eggs to be laid. Using receptor null mutations and cell-specific knockdowns, we found that serotonin signaling through either SER-1/Gαq or SER-7/Gαs alone does not induce egg laying, but these subthreshold signals can combine to promote egg laying. However, using designer receptors or optogenetics to artificially induce high levels of either Gαq signaling or Gαs signaling in the muscles was sufficient to induce egg laying. Conversely, knocking down both Gαq and Gαs in the egg-laying muscle cells induced egg-laying defects stronger than those of a ser-7 ser-1 double knockout. These results suggest that, in the egg-laying muscles, multiple GPCRs for serotonin and other signals each produce weak effects that individually do not result in strong behavioral outcomes. However, they can combine to produce sufficient levels of Gαq and Gαs signaling to promote muscle activity and egg laying.
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