Michaela Nelson

Relative roles of niche and neutral processes in structuring a soil microbial community

Most attempts to identify the processes that structure natural communities have focused on conspicuous macroorganisms whereas the processes responsible for structuring microbial communities remain relatively unknown. Two main theories explaining these processes have emerged; niche theory, which highlights the importance of deterministic processes, and neutral theory, which focuses on stochastic processes. We examined whether neutral or niche-based mechanisms best explain the composition and structure of communities of a functionally important soil microbe, the arbuscular mycorrhizal (AM) fungi. Using molecular techniques, we surveyed AM fungi from 425 individual plants of 28 plant species along a soil pH gradient. There was evidence that both niche and neutral processes structured this community. Species abundances fitted the zero-sum multinomial distribution and there was evidence of dispersal limitation, both indicators of neutral processes. However, we found stronger support that niche differentiation based on abiotic soil factors, primarily pH, was structuring the AM fungal community. Host plant species affected AM fungal community composition negligibly compared to soil pH. We conclude that although niche partitioning was the primary mechanism regulating the composition and diversity of natural AM fungal communities, these communities are also influenced by stochastic-neutral processes. This study represents one of the most comprehensive investigations of community-level processes acting on soil microbes; revealing a community that although influenced by stochastic processes, still responded in a predictable manner to a major abiotic niche axis, soil pH. The strong response to environmental factors of this community highlights the susceptibility of soil microbes to environmental change.

Distinct seasonal assemblages of arbuscular mycorrhizal fungi revealed by massively parallel pyrosequencing

• Understanding the dynamics of rhizosphere microbial communities is essential for predicting future ecosystem function, yet most research focuses on either spatial or temporal processes, ignoring combined spatio-temporal effects. • Using pyrosequencing, we examined the spatio-temporal dynamics of a functionally important community of rhizosphere microbes, the arbuscular mycorrhizal (AM) fungi. We sampled AM fungi from plant roots growing in a temperate grassland in a spatially explicit manner throughout a year. • Ordination analysis of the AM fungal assemblages revealed significant temporal changes in composition and structure. Alpha and beta diversity tended to be negatively correlated with the climate variables temperature and sunshine hours. Higher alpha diversity during colder periods probably reflects more even competitive interactions among AM fungal species under limited carbon availability, a conclusion supported by analysis of beta diversity which highlights how resource limitation may change localized spatial dynamics. • Results reveal distinct AM fungal assemblages in winter and summer at this grassland site. A seasonally changing supply of host-plant carbon, reflecting changes in temperature and sunshine hours, may be the driving force in regulating the temporal dynamics of AM fungal communities. Climate change effects on seasonal temperatures may therefore substantially alter future AM fungal community dynamics and ecosystem functioning.

Molecular phylogeny of choanoflagellates, the sister group to Metazoa

Choanoflagellates are single-celled aquatic flagellates with a unique morphology consisting of a cell with a single flagellum surrounded by a “collar” of microvilli. They have long interested evolutionary biologists because of their striking resemblance to the collared cells (choanocytes) of sponges. Molecular phylogeny has confirmed a close relationship between choanoflagellates and Metazoa, and the first choanoflagellate genome sequence has recently been published. However, molecular phylogenetic studies within choanoflagellates are still extremely limited. Thus, little is known about choanoflagellate evolution or the exact nature of the relationship between choanoflagellates and Metazoa. We have sequenced four genes from a broad sampling of the morphological diversity of choanoflagellates including most species currently available in culture. Phylogenetic analyses of these sequences, alone and in combination, reject much of the traditional taxonomy of the group. The molecular data also strongly support choanoflagellate monophyly rejecting proposals that Metazoa were derived from a true choanoflagellate ancestor. Mapping of a complementary matrix of morphological and ecological traits onto the phylogeny allows a reinterpretation of choanoflagellate character evolution and predicts the nature of their last common ancestor.

Local Adaptation to Soil Hypoxia Determines the Structure of an Arbuscular Mycorrhizal Fungal Community in Roots from Natural CO2 Springs

ABSTRACT The processes responsible for producing and maintaining the diversity of natural arbuscular mycorrhizal (AM) fungal communities remain largely unknown. We used natural CO2 springs (mofettes), which create hypoxic soil environments, to determine whether a long-term, directional, abiotic selection pressure could change AM fungal community structure and drive the selection of particular AM fungal phylotypes. We explored whether those phylotypes that appear exclusively in hypoxic soils are local specialists or widespread generalists able to tolerate a range of soil conditions. AM fungal community composition was characterized by cloning, restriction fragment length polymorphism typing, and the sequencing of small subunit rRNA genes from roots of four plant species growing at high (hypoxic) and low (control) geological CO2 exposure. We found significant levels of AM fungal community turnover (β diversity) between soil types and the numerical dominance of two AM fungal phylotypes in hypoxic soils. Our results strongly suggest that direct environmental selection acting on AM fungi is a major factor regulating AM fungal communities and their phylogeographic patterns. Consequently, some AM fungi are more strongly associated with local variations in the soil environment than with their host plants distribution.

The sodium channel β1 subunit mediates outgrowth of neurite-like processes on breast cancer cells and promotes tumour growth and metastasis

Voltage‐gated Na+ channels (VGSCs) are heteromeric proteins composed of pore‐forming α subunits and smaller β subunits. The β subunits are multifunctional channel modulators and are members of the immunoglobulin superfamily of cell adhesion molecules (CAMs). β1, encoded by SCN1B, is best characterized in the central nervous system (CNS), where it plays a critical role in regulating electrical excitability, neurite outgrowth and migration during development. β1 is also expressed in breast cancer (BCa) cell lines, where it regulates adhesion and migration in vitro. In the present study, we found that SCN1B mRNA/β1 protein were up‐regulated in BCa specimens, compared with normal breast tissue. β1 upregulation substantially increased tumour growth and metastasis in a xenograft model of BCa. β1 over‐expression also increased vascularization and reduced apoptosis in the primary tumours, and β1 over‐expressing tumour cells had an elongate morphology. In vitro, β1 potentiated outgrowth of processes from BCa cells co‐cultured with fibroblasts, via trans‐homophilic adhesion. β1‐mediated process outgrowth in BCa cells required the presence and activity of fyn kinase, and Na+ current, thus replicating the mechanism by which β1 regulates neurite outgrowth in CNS neurons. We conclude that when present in breast tumours, β1 enhances pathological growth and cellular dissemination. This study is the first demonstration of a functional role for β1 in tumour growth and metastasis in vivo. We propose that β1 warrants further study as a potential biomarker and targeting β1‐mediated adhesion interactions may have value as a novel anti‐cancer therapy.

Differential gene expression according to race and host plant in the pea aphid

Host‐race formation in phytophagous insects is thought to provide the opportunity for local adaptation and subsequent ecological speciation. Studying gene expression differences amongst host races may help to identify phenotypes under (or resulting from) divergent selection and their genetic, molecular and physiological bases. The pea aphid (Acyrthosiphon pisum) comprises host races specializing on numerous plants in the Fabaceae and provides a unique system for examining the early stages of diversification along a gradient of genetic and associated adaptive divergence. In this study, we examine transcriptome‐wide gene expression both in response to environment and across pea aphid races selected to cover the range of genetic divergence reported in this species complex. We identify changes in expression in response to host plant, indicating the importance of gene expression in aphid–plant interactions. Races can be distinguished on the basis of gene expression, and higher numbers of differentially expressed genes are apparent between more divergent races; these expression differences between host races may result from genetic drift and reproductive isolation and possibly divergent selection. Expression differences related to plant adaptation include a subset of chemosensory and salivary genes. Genes showing expression changes in response to host plant do not make up a large portion of between‐race expression differences, providing confirmation of previous studies’ findings that genes involved in expression differences between diverging populations or species are not necessarily those showing initial plasticity in the face of environmental change.

Nav1.5 regulates breast tumor growth and metastatic dissemination in vivo.

Voltage-gated Na+ channels (VGSCs) mediate action potential firing and regulate adhesion and migration in excitable cells. VGSCs are also expressed in cancer cells. In metastatic breast cancer (BCa) cells, the Nav1.5 α subunit potentiates migration and invasion. In addition, the VGSC-inhibiting antiepileptic drug phenytoin inhibits tumor growth and metastasis. However, the functional activity of Nav1.5 and its specific contribution to tumor progression in vivo has not been delineated. Here, we found that Nav1.5 is up-regulated at the protein level in BCa compared with matched normal breast tissue. Na+ current, reversibly blocked by tetrodotoxin, was retained in cancer cells in tumor tissue slices, thus directly confirming functional VGSC activity in vivo. Stable down-regulation of Nav1.5 expression significantly reduced tumor growth, local invasion into surrounding tissue, and metastasis to liver, lungs and spleen in an orthotopic BCa model. Nav1.5 down-regulation had no effect on cell proliferation or angiogenesis within the in tumors, but increased apoptosis. In vitro, Nav1.5 down-regulation altered cell morphology and reduced CD44 expression, suggesting that VGSC activity may regulate cellular invasion via the CD44-src-cortactin signaling axis. We conclude that Nav1.5 is functionally active in cancer cells in breast tumors, enhancing growth and metastatic dissemination. These findings support the notion that compounds targeting Nav1.5 may be useful for reducing metastasis.

Three Families of LTR Retrotransposons are Present in the Genome of the Choanoflagellate Monosiga brevicollis

The choanoflagellates are a ubiquitous group of nanoflagellates and the sister group of Metazoa. Examination of the initial draft version of the first choanoflagellate genome, that of Monosiga brevicollis, reveals the presence of three novel families of long terminal repeat (LTR) retrotransposons and an apparent absence of non-LTR retrotransposons and transposons. One of the newly discovered LTR families falls in the chromovirus clade of the Ty3/gypsy group while the other two families are closely related members of the Ty1/copia group. Examination of EST sequences and nucleotide analyses show that all three families are transcriptionally active and potentially functional within the genome of M. brevicollis.

IK channel activation increases tumor growth and induces differential behavioral responses in two breast epithelial cell lines

Many potassium channel families are over-expressed in cancer, but their mechanistic role in disease progression is poorly understood. Potassium channels modulate membrane potential (Vmem) and thereby influence calcium ion dynamics and other voltage-sensitive signaling mechanisms, potentially acting as transcriptional regulators. This study investigated the differential response to over-expression and activation of a cancer-associated potassium channel, the intermediate conductance calcium-activated potassium channel (IK), on aggressive behaviors in mammary epithelial and breast cancer cell lines. IK was over-expressed in the highly metastatic breast cancer cell line MDA-MB-231 and the spontaneously immortalized breast epithelial cell line MCF-10A, and the effect on cancer-associated behaviors was assessed. IK over-expression increased primary tumor growth and metastasis of MDA-MB-231 in orthotopic xenografts, demonstrating for the first time in any cancer type that increased IK is sufficient to promote cancer aggression. The primary tumors had similar vascularization as determined by CD31 staining and similar histological characteristics. Interestingly, despite the increased in vivo growth and metastasis, neither IK over-expression nor activation with agonist had a significant effect on MDA-MB-231 proliferation, invasion, or migration in vitro. In contrast, IK decreased MCF-10A proliferation and invasion through Matrigel but had no effect on migration in a scratch-wound assay. We conclude that IK activity is sufficient to promote cell aggression in vivo. Our data provide novel evidence supporting IK and downstream signaling networks as potential targets for cancer therapies.

Abstract 1972: The sodium channel auxiliary subunit SCN1B promotes breast tumor growth and metastasis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Voltage-gated Na+ channels (VGSCs) are heteromeric proteins composed of pore-forming α subunits and smaller β subunits. The β subunits are multifunctional channel modulators and are members of the immunoglobulin superfamily of cell adhesion molecules (CAMs). β1, encoded by SCN1B, is best characterized in the central nervous system (CNS), where it plays a critical role in regulating electrical excitability, neurite outgrowth and migration during development. β1 is also expressed in breast cancer cell lines, where it regulates adhesion and migration in vitro. Using the Oncomine database, we found that SCN1B mRNA was up-regulated in breast cancer, compared with normal breast. Similarly, β1 protein was up-regulated in a cohort of breast cancer tissue specimens compared with surrounding normal tissue. Over-expression of β1 in MDA-MB-231 cells substantially increased tumor growth and metastasis from orthotopic xenografts in immune-deficient mice. β1 had no effect on the density of Ki67-positive cycling cells in the primary tumors. However, β1 increased the density of CD31-positive vessel structures and reduced the density of apoptotic cells expressing activated caspase-3. β1-expressing MDA-MB-231 cells had an elongate morphology in tumors in vivo and in 2D culture in vitro. β1 potentiated the outgrowth of processes from MDA-MB-231 cells and MCF-7 cells co-cultured with fibroblasts, via trans-homophilic adhesion. Both the src family tyrosine kinase inhibitor PP2 and siRNA targeting fyn kinase blocked β1-mediated process extension in breast cancer cells. Thus, β1-mediated process outgrowth in breast cancer cells requires the presence and activity of fyn kinase. This recapitulates the mechanism by which β1 regulates neurite outgrowth in CNS neurons. We conclude that when present in breast tumors, β1 drives pathological growth and cellular dissemination by recapitulating its well-defined role in CNS ontogeny. This study is the first demonstration of a functional role for β1 as a CAM in tumor growth and metastasis. We propose that targeting β1-mediated adhesion interactions may have potential as a novel anti-cancer therapy. Citation Format: Michaela Nelson, Rebecca Millican-Slater, Lorna C. Forrest, William J. Brackenbury. The sodium channel auxiliary subunit SCN1B promotes breast tumor growth and metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1972. doi:10.1158/1538-7445.AM2014-1972