Evolutionary history and patterns of geographical variation, fertility, and hybridization in Stuckenia (Potamogetonaceae).

Aquatic plant species are often widespread, even across continents. They pose a challenge to species delimitation and taxonomy due to their reduced morphology and high phenotypic plasticity. These difficulties are even more pronounced in the case of interspecific hybridization. We investigate the aquatic plant genus Stuckenia for the first time on a worldwide scale. Expert species determination is aided by sequencing of nuclear ribosomal ITS and 5S-NTS regions and the plastid intergenic spacers rpl20-5'rps12 and trnT-trnL. Nuclear markers are used to infer hybridization, and the maternal origin of hybrids is addressed with plastid markers. Pure species are subjected to phylogenetic analyses. Two main Stuckenia lineages are found: one consists of S. amblyphylla, S. filiformis, S. pamirica, and S. vaginata, the other includes S. pectinata and S. striata. The widespread species S. pectinata, S. filiformis, and S. vaginata show intraspecific genetic variation, which is structured geographically. Many intraspecific hybrids, which are usually fertile, occur between those genotypes. Interspecific hybrids, which are consistently sterile, are detected among all widespread species; some are reported for the first time in several countries and regions. They originated multiple times from reciprocal crosses and reflect the geographical origins of parental genotypes. Intraspecific genetic variation can be higher than interspecific differences between closely related species. Comparison of phenotypic variation in the field and in cultivation with genotypic variation shows that numerous conspicuous forms have been overestimated taxonomically. These are resolved as phenotypes responding to unusual environments, have recurrently evolved adaptations, or represent extreme forms of continuous variation of the recognized species. However, some specific regional lineages, which have evolved from variable species, may be interpreted as early steps of the speciation process. Hybridization has been underestimated in some regions as a source of Stuckenia diversity, and the respective hybrid plants have been misidentified as intraspecific taxa or even as separate species. Many erroneous entries in sequence databases are detected and summarized. This work provides a sound basis for species delimitation and hybrid recognition in this difficult genus.

A Panicum-derived chromosomal segment captured by Hordeum a few million years ago preserves a set of stress-related genes.

Intra-specific variability is a cornerstone of evolutionary success of species. Acquiring genetic material from distant sources is an important adaptive mechanism in bacteria, but it can also play a role in eukaryotes. In this paper, we investigate the nature and evolution of a chromosomal segment of panicoid (Poaceae, Panicoideae) origin occurring in the nuclear genomes of species of the barley genus Hordeum (Pooideae). The segment, spanning over 440 kb in the Asian Hordeum bogdanii and 219 kb in the South American Hordeum pubiflorum, resides on a pair of nucleolar organizer region (NOR)-bearing chromosomes. Conserved synteny and micro-collinearity of the segment in both species indicate a common origin of the segment, which was acquired before the split of the respective barley lineages 5-1.7 million years ago. A major part of the foreign DNA consists of several approximately 68 kb long repeated blocks containing five stress-related protein-coding genes and transposable elements (TEs). Whereas outside these repeats, the locus was invaded by multiple TEs from the host genome, the repeated blocks are rather intact and appear to be preserved. The protein-coding genes remained partly functional, as indicated by conserved reading frames, a low amount of non-synonymous mutations, and expression of mRNA. A screen across Hordeum species targeting the panicoid protein-coding genes revealed the presence of the genes in all species of the section Stenostachys. In summary, our study shows that grass genomes can contain large genomic segments obtained from distantly related species. These segments usually remain undetected, but they may play an important role in the evolution and adaptation of species.

The actin regulator profilin 1 is functionally associated with the mammalian centrosome.

Profilin 1 is a crucial actin regulator, interacting with monomeric actin and several actin-binding proteins controlling actin polymerization. Recently, it has become evident that this profilin isoform associates with microtubules via formins and interferes with microtubule elongation at the cell periphery. Recruitment of microtubule-associated profilin upon extensive actin polymerizations, for example, at the cell edge, enhances microtubule growth, indicating that profilin contributes to the coordination of actin and microtubule organization. Here, we provide further evidence for the profilin-microtubule connection by demonstrating that it also functions in centrosomes where it impacts on microtubule nucleation.

A Fluorophore Fusion Construct of Human Profilin I with Non-Compromised Poly(L-Proline) Binding Capacity Suitable for Imaging.

Profilin is vital for actin organisation in eukaryotic cells. It controls actin filament formation by binding monomeric actin and numerous proteins involved in polarised actin assembly. Important for the latter is the interaction surface formed by the N- and C-terminal helices, which pack close to each other on one side of the molecule at a distance from the actin site and mediate binding to poly-proline sequences present in many of the targeted proteins. Via these interactions, profilin contributes to the spatiotemporal control of actin filament growth. Studies of profilin dynamics in living cells by imaging techniques have been hampered by problems to generate fusion constructs with fluorophore proteins without negatively impacting on its poly-proline binding. With the object to circumvent this problem, we have generated an internal fusion of profilin with the green fluorescent variant citrine, here referred to as citrine-profilin. The characterisation of citrine-profilin (CIT-Pfn) demonstrates that it has full capacity to interact with poly-proline and also binds phosphatidylinositol lipids and actin, albeit with 10 times reduced affinity for the latter. Imaging of living cells expressing CIT-Pfn showed a distribution of the fusion protein similar to endogenous profilin. Furthermore, CIT-Pfn rescued the phenotypes observed after the Crispr/Cas9 knockout of the profilin 1 gene, including the lost migratory capacity characterising the knockout cells. Based on this, we conclude that the CIT-Pfn construct will be useful as a tool for displaying profilin localisation in living cells and obtaining information on its dynamic organisation under different conditions and activations of the actin microfilament and microtubule systems.

Profilin connects actin assembly with microtubule dynamics.

Profilin controls actin nucleation and assembly processes in eukaryotic cells. Actin nucleation and elongation promoting factors (NEPFs) such as Ena/VASP, formins, and WASP-family proteins recruit profilin:actin for filament formation. Some of these are found to be microtubule associated, making actin polymerization from microtubule-associated platforms possible. Microtubules are implicated in focal adhesion turnover, cell polarity establishment, and migration, illustrating the coupling between actin and microtubule systems. Here we demonstrate that profilin is functionally linked to microtubules with formins and point to formins as major mediators of this association. To reach this conclusion, we combined different fluorescence microscopy techniques, including superresolution microscopy, with siRNA modulation of profilin expression and drug treatments to interfere with actin dynamics. Our studies show that profilin dynamically associates with microtubules and this fraction of profilin contributes to balance actin assembly during homeostatic cell growth and affects micro-tubule dynamics. Hence profilin functions as a regulator of microtubule (+)-end turnover in addition to being an actin control element.

Involvement of MAP kinases in the cytotoxicity of acyclic nucleoside phosphonates.

BACKGROUND: 9-[2-(phosphonomethoxy)ethyl] guanine (PMEG) is a nucleotide analogue with anticancer activity. Here we investigate the role of ERK, p38, JNK and AKT kinases in PMEG-induced apoptosis. MATERIALS AND METHODS: CCRF-CEM and HL-60 leukemia cells were used to assess MAPK mRNA and protein expression in PMEG-treated cells. MAPK activation was measured using phospho-specific antibodies. Apoptosis was evaluated by caspase-3 and PARP cleavage. RESULTS: Up-regulation of p38ß, γ and δ mRNA were observed following PMEG treatment of CCRF-CEM cells, however, the total protein expression remained unchanged. Neither PMEG nor its analogue 9-[2-(phosphonomethoxy) ethyl]-2,6-diaminopurine (PMEDAP) induced p38 kinase phosphorylation in CCRF-CEM cells, whereas increased p38 phosphorylation was observed in HL-60 cells. The ERK pathway was also activated by these compounds. Pretreatment of the cells with the p38 inhibitor SB203580 diminished drug-induced apoptosis whereas inhibition of ERK, JNK or AKT pathways did not. [corrected]. CONCLUSION: PMEG- and PMEDAP-induced. [corrected].