Congruence and conflict: case studies of morphotaxonomy versus rDNA gene tree phylogeny among articulate brachiopods (Brachiopoda: Rhynchonelliformea), with description of a new genus

We present five case studies among articulate (rhynchonelliform) brachiopods, i.e. of Rhynchonellida, Cancellothyridoidea, Terebratuloidea, Dyscolioidea, Laqueoidea, and various terebratulids with modified long‐loops, in an attempt to illustrate and better understand congruence and conflict between morpho‐classification and rDNA‐based molecular clade structure, having been prompted to address these issues by difficulties encountered when describing the newly collected brachiopod, E biscothyris bellonensis gen. et sp. nov. The five studies reveal dramatic conflict in the Rhynchonellida and Terebratuloidea/Dyscolioidea, good congruence in the Cancellothyridoidea and Laqueoidea, and fair congruence (albeit with weak phylogenetic signal) in the long‐looped terebratulids. We suggest that the leading cause of the observed conflict lies in the use of inadequately specific morphological characters and morpho‐classification. Phylogenetic systematic (cladistic) analyses of Rhynchonellida also conflict markedly with the rDNA gene tree, leading us to recognize that such analyses are not only conceptually circular (using morphological characters to assess a morphological classification) but also to propose that they are biased by the act of classification that necessarily precedes the identification of putatively homologous characters; when the prior classification does not reflect evolutionary history, phylogenetic analysis will do likewise. In addition, we propose that the brachiopod community has overlooked the significance of two sources of morphological homoplasy affecting brachiopod systematics: (1) the loss of co‐adapted genomic complexes caused by mass extinctions at the end of the Permian; and (2) the pervasive consequences of developmental integration and constraint resulting from the integrated roles of the outer mantle epithelium in shell deposition and growth that underly the determination of form and the shell‐based classification. © 2015 The Linnean Society of London     

Topology of Endoplasmic Reticulum‐Associated Cellular and Viral Proteins Determined with Split‐GFP

The split green fluorescent protein (GFP) system was adapted for investigation of the topology of ER‐associated proteins. A 215‐amino acid fragment of GFP (S1–10) was expressed in the cytoplasm as a free protein or fused to the N‐terminus of calnexin and in the ER as an intraluminal protein or fused to the C‐terminus of calnexin. A 16‐amino acid fragment of GFP (S11) was fused to the N‐ or C‐terminus of the target protein. Fluorescence occurred when both GFP fragments were in the same intracellular compartment. After validation with the cellular proteins PDI and tapasin, we investigated two vaccinia virus proteins (L2 and A30.5) of unknown topology that localize to the ER and are required for assembly of the viral membrane. Our results indicated that the N‐ and C‐termini of L2 faced the cytoplasmic and luminal sides of the ER, respectively. In contrast both the N‐ and C‐termini of A30.5 faced the cytoplasm. The system offers advantages for quickly determining the topology of intracellular proteins: the S11 tag is similar in length to commonly used epitope tags; multiple options are available for detecting fluorescence in live or fixed cells; transfection protocols are adaptable to numerous expression systems and can enable high throughput applications.      

Internalization of staphylococcal leukotoxins that bind and divert the C5a receptor is required for intracellular Ca2+ mobilization by human neutrophils

A growing number of receptors, often associated with the innate immune response, are being identified as targets for bacterial toxins of the beta‐stranded pore‐forming family. These findings raise the new question of whether the receptors are activated or merely used as docking points facilitating the formation of a pore. To elucidate whether the Staphylococcus aureus Panton‐Valentine leukocidin and the leukotoxin HlgC/HlgB act through the C5a receptor (C5aR) as agonists, antagonists or differ from the C5a complement‐derived peptide, their activity is explored on C5aR‐expressing cells. Both leukotoxins equally bound C5aR in neutrophils and in stable transfected U937 cells and initiated mobilization of intracellular Ca²⁺. HlgC/HlgB requires the presence of robust intracellular acidic Ca²⁺ stores in order to evoke a rise in free [Ca²⁺]_i, while the LukS‐PV/LukF‐PV directly altered reticular Ca²⁺ stores. Intracellular target specificity is conferred by the F‐subunit associated to the S‐subunit binding the receptor. Furthermore, internalization of the two leukotoxin components (S‐ and F‐subunits) associated to C5aR is required for the initiation of [Ca²⁺]_i mobilization. Electrophysiological recordings on living cells demonstrated that LukS‐PV/LukF‐PV does not alter the membrane resistance of C5aR‐expressing cells. The present observations suggest that part of the pore‐forming process occurs in distinct intracellular compartments rather than at the plasma membrane.