Search Results (3)

Background/Objectives: The Structural Maintenance of Chromosomes (SMC) proteins form essential heterocomplexes for the preservation of DNA structure and its functions, and hence cell viability. The SMC5/6 dimer is assembled by direct interactions of ATP heads via the kleisin NSE4 bridge and by SMC hinges. The structure might be interrupted by a single point mutation within a conserved motif of the SMC6-hinge. We describe the phenomena associated with the impairment of the SMC5/6 complex with morphology, repair of DNA double strand breaks (DSB), mutagenesis, recombination and gene targeting (GT) in the moss Physcomitrium patens (P. patens). Methods: Using CRISPR/Cas9-directed oligonucleotide replacement, we have introduced two close G to R point mutations in the hinge domain of SMC6 of P. patens and show that both mutations are not toxic and allow viability of mutant lines. Results: The G514R mutation fully prevents the interaction of SMC6 not only with SMC5, but also with NSE5 and NSE6, while the mutation at G517R has no effect. The Ppsmc6_G514R line has aberrant morphology, spontaneous and bleomycin-induced mutagenesis, and maintenance of the number of rDNA copies. The most unique feature is the interference with gene targeting (GT), which is completely abolished. In contrast, the Ppsmc6_G517R line is close to WT in many aspects. Surprisingly, both mutations have no direct effect on the rate of DSB repair in dividing and differentiated cells. Conclusions: Abolished interactions of SMC6 with SMC5 and NSE5,6 partners, which allow DSB repair, but impair other repair and recombination functions, suggests also regulatory role for SMC6. Full article

Gravi-morphoses affect the variability of plants and are the morphogenetic adaptation to different environmental conditions. Gravity-dependent phenotypic plasticity of gametophytes as well as gravi-sensitivity of moss protonemata in microgravity and simulated microgravity conditions are discussed. The moss protonema, a filamentous multicellular system, representing a juvenile stage of moss development, develops as a result of the elongation and division of the apical cell. This apical cell of the protonema is a unique object for research on moss gravi-sensitivity, as graviperception and gravitropic growth occur within the same single cell. Attention is focused on the influence of gravity on bryophyte ontogenesis, including the gravitropic reactivity of moss protonemata, gravi-sensitivity at the stage of leafy shoot development and sporogonium formation, gravity-influenced morphogenesis of apical cell budding, and gravity-dependent spiral growth patterns. The role of gravireceptors in the growth processes of mosses at the cellular level under microgravity conditions are being discussed, as well as the involvement of auxin transport, Ca2+-induced gravitropism and the cytoskeleton in gravitropic reactions. Full article

In conventional light microscopy, the adjacent cell walls of filamentous moss protonemata are seen from its narrow side thereby obscuring the major area of cell–cell connection. Optical sectioning, segmentation and 3D reconstructions allow the tilting and rotation of intracellular structures thereby greatly improving our understanding of interaction between organelles, membranes and the cell wall. Often, the findings also allow for conclusions on the respective functions. The moss Physcomitrium (Physcomitrella) patens is a model organism for growth, development and morphogenesis. Its filamentous protonemata are ideal objects for microscopy. Here, we investigated the cell wall between two neighboring cells and the connection of membranes towards this wall after plasmolysis in 0.8 M mannitol. An m-green fluorescent protein (GFP)-HDEL cell line was used to visualize the endoplasmatic reticulum (ER), the plasma membrane (PM) was stained with FM4-64. Our studies clearly show the importance of cell–cell contacts in P. patens protonemata. In 86% of the investigated cell pairs, at least one of the protoplasts remained fully attached to the adjacent cell wall. By tilting of z-stacks, volume renderings and 3D reconstructions, we visualized the amount of attached/detached PM and ER components after plasmolysis and membrane piercings through the wall of cell neighbors. Full article