We developed a method to produce discrete fibrin microthreads, which can be seeded with human mesenchymal stem cells (hMSCs) and used as a suture to enhance the efficiency and localization of cell delivery. To assess the efficacy of fibrin microthreads to support hMSC attachment, proliferation and survival, microthreads (100 µm diameter per microthread) were bundled together, seeded with 50,000 hMSCs for 2 hours, and cultured for 5 days. Cell density on microthread bundles increased over time in culture, to a maximum average density of 731±101 cells/mm 2 after 5 days. A LIVE/DEAD assay confirmed that the cells were viable and Ki-67 staining verified hMSC proliferation. Additionally, functional differentiation assays demonstrated that hMSCs cultured on microthreads retained their ability to differentiate into adipocytes and osteocytes. The results of this study demonstrate that fibrin microthreads support hMSC viability and proliferation, while maintaining their multipotency. We anticipate that these cell-seeded fibrin microthreads will serve a platform technology to improve localized delivery and engraftment of viable cells to damaged tissue.

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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 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.

Generation of double-stranded RNA targeting the adenine phosphoribosyltransferase gene in tandem with a target gene enables positive selection of strongly silencing plants.

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...

RNA interference (RNAi) enables flexible and dynamic interrogation of entire gene families or essential genes without the need for exogenous proteins, unlike CRISPR-Cas technology. Unfortunately, isolation of plants undergoing potent gene silencing requires laborious design, visual screening, and physical separation for downstream characterization. Here, we developed a novel APT-based RNAi technology (APTi) in Physcomitrella patens that simultaneously improves upon the multiple limitations of current RNAi techniques. APTi exploits the pro-survival output of transiently silencing the APT gene in the presence of 2-fluoradenine, thereby establishing survival itself as a reporter of RNAi. To maximize silencing efficacy of gene targets we created vectors that facilitate insertion of any gene target sequence in tandem with the APT silencing motif. The APTi approach resulted in a homogenous population of P. patens mutants specific for our gene target, with zero surviving background plants within 8 days. The observed mutants directly corresponded to a maximal 93% reduction of the tested target protein, substantially exceeding previous dsRNA methods. The positive selection nature of APTi represents a fundamental improvement in RNAi technology and will contribute to the growing demand for technologies amenable to high-throughput phenotyping.One-sentence summaryGeneration of dsRNA targeting the APT gene in tandem with a target gene enables positive selection of strongly silencing plants.