Serotonin transporter oligomerization documented in RN46A cells and neurons by sensitized acceptor emission FRET and fluorescence lifetime imaging microscopy

The serotonin transporter is a member of the monoamine transporter family that also includes transporters of dopamine and norepinephrine. We have used sensitized acceptor emission fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) to study the oligomerization of SERT in HEK-MSR-239 cells, RN46A cells and in cultured hippocampal neurons. We were able to show identical FRET efficiencies in cell lines as well as in primary cultured hippocampal neurons, demonstrating that the oligomerization is cell type independent. The results obtained with both FRET approaches are very similar and furthermore, in agreement with previous results obtained by donor bleaching FRET microscopy.     

Freeze-preservation of buds from Scots pine trees

A procedure has been developed for freeze-preservation of buds of the Scots pine (Pinus sylvestris L.). Instead of liquid nitrogen, cold storage in –80°C was used. The partly dormant material used in the experiments was obtained directly from a natural stand in Northern Finland and no prefreezing or cryoprotectants for preconditioning were used. Cooling velocity was 1°C/min up to a terminal freezing temperature of –39°C, after which the buds were immersed in liquid nitrogen at –196°C for 10 minutes. The material was then transferred to a deepfreezer at –80°C and stored up to 6 months. After rapid thawing, the buds were sterilized and their viability was tested by FDA staining and by culturing meristems on 1/2 MS medium for at least two weeks. All the freezing experiments were performed during March and April. The best survival of buds (90–100%) was achieved at the beginning of April, after which a pronounced decline in survival occurred obviously due to a rise in the water content of the buds.     

Sipunculan larvae and "cosmopolitan" species

Sipuncula is a relatively small taxon with roughly 150 recognized species. Many species are geographically widespread or "cosmopolitan." The pelagosphera larvae of some species are estimated to spend several months in the plankton. However, recent molecular evidence suggests that many of the "cosmopolitan" species actually represent species-complexes, some not even monophyletic. Herein, we present data on three sipunculan species with different developmental modes that occur both in the Sea of Japan and in the Northeast Pacific. The development of the three species-Phascolosoma agassizii, Thysanocardia nigra, and Themiste pyroides-is exceptionally well studied in both regions of the Pacific. Significant differences have been observed between the two regions with respect to egg size, developmental mode, and developmental timing. In general, eggs are larger and development slower in the Northeast Pacific when compared with the Sea of Japan. These differences have been explained as a result of phenotypic plasticity exhibited under different environmental conditions, in particular temperature, but we show that the populations of all three species are also remarkably distinct genetically and that gene flow between the two regions is extremely unlikely. In Thysanocardia nigra, we even found two very distinct genetic lineages within the same location in the Northeast Pacific. The amount of genetic divergence between populations from the Sea of Japan and those from the Northeast Pacific is not correlated with developmental mode. Themiste pyroides, the species with the most abbreviated development, actually has the least degree of genetic divergence between the regions. Analyses of molecular variance show that the majority of the observed variation in all three species is between the regions. We conclude that all three "cosmopolitan" species actually represent complexes of cryptic or pseudo-cryptic species. These examples demonstrate that a solid taxonomic framework based on molecular and morphological evidence is a prerequisite for evaluating relationships between dispersal capabilities, species' ranges, and the connectivity of populations.     

Distinct requirement for an intact dimer interface in wild-type, V600E and kinase-dead B-Raf signalling

The dimerisation of Raf kinases involves a central cluster within the kinase domain, the dimer interface (DIF). Yet, the importance of the DIF for the signalling potential of wild-type B-Raf (B-Raf(wt)) and its oncogenic counterparts remains unknown. Here, we show that the DIF plays a pivotal role for the activity of B-Raf(wt) and several of its gain-of-function (g-o-f) mutants. In contrast, the B-Raf(V600E), B-Raf(insT) and B-Raf(G469A) oncoproteins are remarkably resistant to mutations in the DIF. However, compared with B-Raf(wt), B-Raf(V600E) displays extended protomer contacts, increased homodimerisation and incorporation into larger protein complexes. In contrast, B-Raf(wt) and Raf-1(wt) mediated signalling triggered by oncogenic Ras as well as the paradoxical activation of Raf-1 by kinase-inactivated B-Raf require an intact DIF. Surprisingly, the B-Raf DIF is not required for dimerisation between Raf-1 and B-Raf, which was inactivated by the D594A mutation, sorafenib or PLX4720. This suggests that paradoxical MEK/ERK activation represents a two-step mechanism consisting of dimerisation and DIF-dependent transactivation. Our data further implicate the Raf DIF as a potential target against Ras-driven Raf-mediated (paradoxical) ERK activation.     

Actin depolymerizing factors ADF7 and ADF10 play distinct roles during pollen development and pollen tube growth

An important player in actin remodeling is the actin depolymerizing factor (ADF) which increases actin filament treadmilling rates. Previously, we had prepared fluorescent protein fusions of two Arabidopsis pollen specific ADFs, ADF7 and ADF10. These had enabled us to determine the temporal expression patterns and subcellular localization of these proteins during male gametophyte development. Here we generated stable transformants containing both chimeric genes allowing for simultaneous imaging and direct comparison. One of the striking differences between the two proteins was the localization profile in the growing pollen tube apex. Whereas ADF10 was associated with the filamentous actin array forming the subapical actin fringe, ADF7 was present in the same cytoplasmic region, but in diffuse form. This suggests that ADF7 is involved in the high actin turnover that is likely to occur in the fringe by continuously and efficiently depolymerizing filamentous actin and supplying monomeric actin to the advancing end of the fringe. The possibility to visualize both of these pollen-specific ADFs simultaneously opens avenues for future research into the regulatory function of actin binding proteins in pollen.     

Karyotype variation of CHO host cell lines over time in culture characterized by chromosome counting and chromosome painting

Genomic rearrangements are a common phenomenon in rapidly growing cell lines such as Chinese hamster ovary (CHO) cells, a feature that in the context of production of biologics may lead to cell line and product instability. Few methods exist to assess such genome wide instability. Here, we use the population distribution of chromosome numbers per cell as well as chromosome painting to quantify the karyotypic variation in several CHO host cell lines. CHO‐S, CHO‐K1 8 mM glutamine, and CHO‐K1 cells adapted to grow in media containing no glutamine were analyzed over up to 6 months in culture. All three cell lines were clearly distinguishable by their chromosome number distribution and by the specific chromosome rearrangements that were present in each population. Chromosome Painting revealed a predominant karyotype for each cell line at the start of the experiment, completed by a large number of variants present in each population. Over time in culture, the predominant karyotype changed for CHO‐S and CHO‐K1, with the diversity increasing and new variants appearing, while CHO‐K1 0 mM Gln preferred chromosome pattern increased in percent of the population over time. As control, Chinese hamster lung fibroblasts were shown to also contain an increasing number of variants over time in culture.     

The curious case of Hermodice carunculata (Annelida: Amphinomidae): evidence for genetic homogeneity throughout the Atlantic Ocean and adjacent basins

Over the last few decades, advances in molecular techniques have led to the detection of strong geographic population structure and cryptic speciation in many benthic marine taxa, even those with long‐lived pelagic larval stages. Polychaete annelids, in particular, generally show a high degree of population divergence, especially in mitochondrial genes. Rarely have molecular studies confirmed the presence of ‘cosmopolitan’ species. The amphinomid polychaete Hermodice carunculata was long considered the sole species within its genus, with a reported distribution throughout the Atlantic and adjacent basins. However, recent studies have indicated morphological differences, primarily in the number of branchial filaments, between the East and West Atlantic populations; these differences were invoked to re‐instate Hermodice nigrolineata, formerly considered a junior synonym of H. carunculata. We utilized sequence data from two mitochondrial (cytochrome c oxidase subunit I, 16S rDNA) markers and one nuclear (internal transcribed spacer) marker to examine the genetic diversity of Hermodice throughout its distribution range in the Atlantic Ocean, including the Mediterranean Sea, the Caribbean Sea, the Gulf of Mexico and the Gulf of Guinea. Our analyses revealed generally low genetic divergences among collecting localities and between the East and West Atlantic, although phylogenetic trees based on mitochondrial data indicate the presence of a private lineage in the Mediterranean Sea. A re‐evaluation of the number of branchial filaments confirmed differences between East and West Atlantic populations; however, the differences were not diagnostic and did not reflect the observed genetic population structure. Rather, we suspect that the number of branchial filaments is a function of oxygen saturation in the environment. Our results do not support the distinction between H. carunculata in the West Atlantic and H. nigrolineata in the East Atlantic. Instead, they re‐affirm the older notion that H. carunculata is a cohesive species with a broad distribution across the Atlantic Ocean.     

Microbial Communities Associated with Zones of Elevated Magnetic Susceptibility in Hydrocarbon-Contaminated Sediments

Recent studies suggest that magnetic susceptibility (MS) measurements can play an important role in identifying zones where microbial-mediated iron mineral transformations are occurring. Here we investigated the microbial community variations within zones of elevated MS in a petroleum hydrocarbon-contaminated aquifer near Bemidji, Minnesota, USA. Our main objective was to 1) identify the key microbial populations that may play a role in hydrocarbon degradation, 2) analyze which microbial populations could be connected to the elevated MS and 3) explore the use of non-destructive geophysical techniques as a tool to guide microbial sampling. Clone libraries based on the 16S rRNA gene revealed the presence of iron-reducing β-Proteobacteria in the vadose zone, whereas the free petroleum phase on the water table was characterized by a methanogenic consortium, in which the syntrophic δ-proteobacterium Smithella and the hydrogenotrophic Methanoregula predominated. Nonmetric multidimensional scaling (NMDS) found a close relationship between elevated MS values and the methanogenic hydrocarbon-degrading consortium. Our results suggest that magnetic susceptibility measurements can guide microbiologists to zones of active microbial biodegradation in aged petroleum spills.     

Targeted resequencing reveals geographical patterns of differentiation for loci implicated in parallel evolution

Parallel divergence and speciation provide evidence for the role of divergent selection in generating biological diversity. Recent studies indicate that parallel phenotypic divergence may not have the same genetic basis in different geographical locations – ‘outlier loci’ (loci potentially affected by divergent selection) are often not shared among parallel instances of phenotypic divergence. However, limited sharing may be due, in part, to technical issues if false‐positive outliers occur. Here, we test this idea in the marine snail Littorina saxatilis, which has evolved two partly isolated ecotypes (adapted to crab predation vs. wave action) in multiple locations independently. We argue that if the low extent of sharing observed in earlier studies in this system is due to sampling effects, we expect outliers not to show elevated F_ST when sequenced in new samples from the original locations and also not to follow predictable geographical patterns of elevated F_ST. Following a hierarchical sampling design (within vs. between country), we applied capture sequencing, targeting outliers from earlier studies and control loci. We found that outliers again showed elevated levels of F_ST in their original location, suggesting they were not generated by sampling effects. Outliers were also likely to show increased F_ST in geographically close locations, which may be explained by higher levels of gene flow or shared ancestral genetic variation compared with more distant locations. However, in contrast to earlier findings, we also found some outlier types to show elevated F_ST in geographically distant locations. We discuss possible explanations for this unexpected result.     

Shedding light on host niches: label‐free in situ detection of Mycobacterium gordonae via carotenoids in macrophages by Raman microspectroscopy

Macrophages are the primary habitat of pathogenic mycobacteria during infections. Current research about the host–pathogen interaction on the cellular level is still going on. The present study proves the potential of Raman microspectroscopy as a label‐free and non‐invasive method to investigate intracellular mycobacteria in situ. Therefore, macrophages were infected with Mycobacterium gordonae, a mycobacterium known to cause inflammation linked to intracellular survival in macrophages. Here, we show that Raman maps provided spatial and spectral information about the position of bacteria within determined cell margins of macrophages in two‐dimensional scans and in three‐dimensional image stacks. Simultaneously, the relative intracellular concentration and distributions of cellular constituents such as DNA, proteins and lipids provided phenotypic information about the infected macrophages. Locations of bacteria outside or close to the outer membrane of the macrophages were notably different in their spectral pattern compared with intracellular once. Furthermore, accumulations of bacteria inside of macrophages exhibit distinct spectral/molecular information because of the chemical composition of the intracellular microenvironment. The data show that the connection of microscopically and chemically gained information provided by Raman microspectroscopy offers a new analytical way to detect and to characterize the mycobacterial infection of macrophages.