Rab-E and its interaction with myosin XI are essential for polarised 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.

Robust Survival-Based RNA Interference of Gene Families Using in Tandem Silencing of Adenine Phosphoribosyltransferase.

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 an adenine phosphoribosyltransferase (APT)-based RNAi technology (APTi) in Physcomitrella patens that improves upon the multiple limitations of current RNAi techniques. APTi exploits the prosurvival output of transiently silencing APT in the presence of 2-fluoroadenine, thereby establishing survival itself as a reporter of RNAi. To maximize the silencing efficacy of gene targets, we created vectors that facilitate insertion of any gene target sequence in tandem with the APT silencing motif. We tested the efficacy of APTi with two gene families, the actin-dependent motor, myosin XI (a,b), and the putative chitin receptor Lyk5 (a,b,c). The APTi approach resulted in a homogenous population of transient P. patens mutants specific for our gene targets with zero surviving background plants within 8 d. The observed mutants directly corresponded to a maximal 93% reduction of myosin XI protein and complete loss of chitin-induced calcium spiking in the Lyk5-RNAi background. 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.

Orchestrating cell morphology from the inside out - using polarized cell expansion in plants as a model.

Intracellular organization forms the basis of changes in the extracellular matrix. In walled cells, these changes are essential for morphogenesis and growth. The highly polarized cells of mosses and liverworts together with root hairs and pollen tubes are geometrically simple cells that develop in the absence of complex tissue-scale signaling, providing an excellent model to study cell polarity. Recent advances present a unifying theme where the cytoskeleton and its associated motors work in coordination with vesicle trafficking. This coordination results in a recycling system near the cell tip, where endocytosed molecules are sorted and combined with exocytic cargo driving growth. Interestingly, functional similarities between filamentous fungi and plants promise to advance our understanding of cell polarization and growth across kingdoms.

Conditional genetic screen in Physcomitrella patens reveals a novel microtubule depolymerizing-end-tracking protein.

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.

Fibrin microthreads support mesenchymal stem cell growth while maintaining differentiation potential.

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 h, 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. In addition, 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 as a platform technology to improve localized delivery and engraftment of viable cells to damaged tissue.