Juvenile survival in a unisexual/sexual complex of mollies

Unisexual species like the gynogenetic Amazon molly, Poecilia formosa, enjoy a twofold advantage over sexual species, because they do not produce males. Therefore, unisexuals should be able to outcompete and consequently, replace sexual species. For sperm-dependent (gynogenetic) unisexuals this creates a paradox: they cannot replace their sexual hosts without eradicating themselves. Thus, mechanisms must be in place to stabilize such mating systems. We assessed juvenile survivorship between asexual P. formosa and sexual Poecilia latipinna as a possible factor allowing for persistence and coexistence between the two sympatric species. Offspring of gynogenetic Amazon mollies did not differ significantly in survivorship compared to their sexual host, the Sailfin molly, P. latipinna. The presence of an adult female significantly reduced survival in both species, suggesting that filial cannibalisms operates in this system, but does not appear to play a role in stabilizing mixed sexual/asexual populations. Clark Hubbs, who spent 59 years at the University of Texas and was widely regarded as one of the state’s foremost researchers in the field of ichthyology, the study of fish, passed away February 3rd of 2008 after a long battle with colorectal cancer. He was 86.     

Characterization of the solution structure of a neuroligin/beta-neurexin complex

Neuroligins are post-synaptic cell adhesion molecules that promote synaptic maturation and stabilization upon binding with pre-synaptic partners, the alpha- and beta-neurexins. Using a combination of analytical ultracentrifugation, small angle X-ray, and neutron scattering, we have characterized the low-resolution three-dimensional structure of the extracellular domain of the neuroligins, free in solution, and in complex with beta-neurexin. The globular extracellular domain of the neuroligins forms stable homodimers through a four-helix bundle typical of the cholinesterases and other members of the alpha/beta-hydrolase fold family. The presence of the stalk region adds to the extracellular domain of neuroligin-1 an elongated structure, suggesting a rod-like nature of the stalk domain. Sedimentation equilibrium coupled with solution scattering data of the beta-neurexin/neuroligin-1 complex indicated a 2:2 stoichiometry where two beta-neurexin molecules bind to a neuroligin-1 dimer. Deuteration of neurexin allowed us to collect neutron scattering data that, in combination with other biochemical techniques, provide a basis for optimizing the positioning of each component in a detailed computational model of the neuroligin/neurexin complex. As several mutations of both neurexin and neuroligin genes have been linked to autism spectrum disorders and mental retardation, these new structures provide an important framework for the study of altered structure and function of these synaptic proteins.     

Light-induced fluorescence quenching in the light-harvesting chlorophyll a/b protein complex

Summary Irradiation of the principal photosystem II light-harvesting chlorophyll-protein antenna complex, LHC II, with high light intensities brings about a pronounced quenching of the chlorophyll fluorescence. Illumination of isolated thylakoids with high light intensities generates the formation of quenching centres within LHC II in vivo, as demonstrated by fluorescence excitation spectroscopy. In the isolated complex it is demonstrated that the light-induced fluorescence quenching: a) shows a partial, biphasic reversibility in the dark; b) is approximately proportional to the light intensity; c) is almost independent of temperature in the range 0–30°C; d) is substantially insensitive to protein modifying reagents and treatments; e) occurs in the absence of oxygen. A possible physiological importance of the phenomenon is discussed in terms of a mechanism capable of dissipating excess excitation energy within the photosystem II antenna.Abbreviations chla chlorophyll a - chlb chlorophyll b - F₀ fluorescence yield with reaction centers open - F_m fluorescence yield with reaction centres closed - F_i fluorescence at the plateau level of the fast induction phase - LHC II light-harvesting chlorophyll a/b protein complex II - PS II photosystem II - PSI photosystem I - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

Evidence for a degradosome-like complex in the mitochondria of Trypanosoma brucei

Mitochondrial RNA turnover in yeast involves the degradosome, composed of DSS-1 exoribonuclease and SUV3 RNA helicase. Here, we describe a degradosome-like complex, containing SUV3 and DSS-1 homologues, in the early branching protozoan, Trypanosoma brucei. TbSUV3 is mitochondrially localized and co-sediments with TbDSS-1 on glycerol gradients. Co-immunoprecipitation demonstrates that TbSUV3 and TbDSS-1 associate in a stable complex, which differs from the yeast degradosome in that it is not stably associated with mitochondrial ribosomes. This is the first report of a mitochondrial degradosome-like complex outside of yeast. Our data indicate an early evolutionary origin for the mitochondrial SUV3/DSS-1 containing complex.

Structured summary

MINT-7187980: SUV3 (genbank_protein_gi:XP_844349) and DSS1 (uniprotkb:Q38EM3) colocalize (MI:0403) by cosedimentation (MI:0027)MINT-7188074: SUV3 (genbank_protein_gi:XP_844349) physically interacts (MI:0914) with DSS1 (uniprotkb:Q38EM3) by anti tag co-immunoprecipitation (MI:0007)     

Crystal structure of a beta-catenin/Tcf complex

The Wnt signaling pathway plays critical roles in embryonic development and tumorigenesis. Stimulation of the Wnt pathway results in the accumulation of a nuclear beta-catenin/Tcf complex, activating Wnt target genes. A crystal structure of beta-catenin bound to the beta-catenin binding domain of Tcf3 (Tcf3-CBD) has been determined. The Tcf3-CBD forms an elongated structure with three binding modules that runs antiparallel to beta-catenin along the positively charged groove formed by the armadillo repeats. Structure-based mutagenesis defines three sites in beta-catenin that are critical for binding the Tcf3-CBD and are differentially involved in binding APC, cadherin, and Axin. The structural and mutagenesis data reveal a potential target for molecular drug design studies.     

Visualization of a DNA-PK/PARP1 complex

The DNA-dependent protein kinase (DNA-PK) and Poly(ADP-ribose) polymerase-1 (PARP1) are critical enzymes that reduce genomic damage caused by DNA lesions. They are both activated by DNA strand breaks generated by physiological and environmental factors, and they have been shown to interact. Here, we report in vivo evidence that DNA-PK and PARP1 are equally necessary for rapid repair. We purified a DNA-PK/PARP1 complex loaded on DNA and performed electron microscopy and single particle analysis on its tetrameric and dimer-of-tetramers forms. By comparison with the DNA-PK holoenzyme and fitting crystallographic structures, we see that the PARP1 density is in close contact with the Ku subunit. Crucially, PARP1 binding elicits substantial conformational changes in the DNA-PK synaptic dimer assembly. Taken together, our data support a functional, in-pathway role for DNA-PK and PARP1 in double-strand break (DSB) repair. We also propose a NHEJ model where protein-protein interactions alter substantially the architecture of DNA-PK dimers at DSBs, to trigger subsequent interactions or enzymatic reactions.     

Endocytosis of the TCR/CD3 complex and the class-I major histocompatibility complex in a human T cell line.

We investigated the expression of the T cell receptor (TCR)/CD3 complex on a CD4-positive human T cell lymphoma cell line treated with phorbol myristate acetate (PMA) and/or CA2+ ionophore using fluorescence flow cytometry and fluorescence microscopic analysis. PMA induced a significant decrease in the expression of the CD3 complex on the cell membranes. Fluorescence microscopy confirmed that the down regulation is due to internalization of the antigens. Ca2+ ionophore treatment had no effect on the internalization of the CD3 complex. Double staining revealed that the vesicles containing the internalized CD3 complex and those containing intra-cytoplasmic class I major histocompatibility complex antigen had similar distribution in the PMA-stimulated cells, implying coexistence of these two antigens in a cytoplasmic perinuclear distribution.     

Coactivator functions in a stoichiometric complex with anaphase-promoting complex/cyclosome to mediate substrate recognition

The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit E3 ligase required for ubiquitin-dependent proteolysis of cell-cycle-regulatory proteins, including mitotic cyclins and securin/Pds1. Regulation of APC/C activity and substrate recognition, mediated by the coactivators Cdc20 and Cdh1, is fundamental to cell-cycle control. However, the precise mechanism by which coactivators stimulate APC/C ubiquitylation activity and the nature of the substrate-binding sites on the activated APC/C are not understood. Here, we show that the optimal interaction of substrate with APC/C is dependent specifically on the simultaneous association of coactivator. This is consistent with a model whereby both core APC/C subunits and coactivators contribute recognition sites for substrates, accounting for the bipartite nature (D and KEN boxes) of most APC/C degradation signals. A direct and stoichiometric function for the coactivators could explain how specific substrates are recognized by APC/C in a cell-cycle-specific manner, and how coactivator stimulates APC/C ubiquitylation activity.     

Conservation of the TRAPPII-specific subunits of a Ypt/Rab exchanger complex

Background  

Ypt/Rab GTPases and their GEF activators regulate intra-cellular trafficking in all eukaryotic cells. In S. cerivisiae, the modular TRAPP complex acts as a GEF for the Golgi gatekeepers: Ypt1 and the functional pair Ypt31/32. While TRAPPI, which acts in early Golgi, is conserved from fungi to animals, not much is known about TRAPPII, which acts in late Golgi and consists of TRAPPI plus three additional subunits.     

Prediction of the tertiary structure of a caspase-9/inhibitor complex

Apoptosis, or programmed cell death, plays a central role in the development and homeostasis of an organism. The breakdown of cellular proteins in apoptosis is mediated by caspases, which comprise a highly conserved family of cysteine proteases with specificity for aspartic acid residues at the P1 positions of their substrates. Multiple lines of evidence show that caspase-9 is critical for an apoptosis pathway mediated via the mitochondria. In this study, the three-dimensional structure of the catalytic domain of caspase-9 and its interaction with the inhibitor acetyl-Asp-Val-Ala-Asp fluoromethyl ketone (Ac-DVAD-fmk) have been predicted by a segment matching modeling procedure. As expected, the predicted caspase-9 structure shows both a high similarity in the overall folding topology and remarkable differences in the surface loop regions as compared to other caspase family members such as caspase-1, -3 and -8, for which crystal structures have been determined. This kind of comparative analysis reflects the convergence-divergence duality among the caspases. Moreover, some subtle differences have been observed between caspase-9 and caspase-3 in the subsite contacts with the covalently linked inhibitor Ac-DVAD-fmk. Based on the X-ray structural analysis of caspase-8, a main chain carbonyl oxygen appears to be involved in a catalytic triad with the active site Cys and His residues. The corresponding carbonyl oxygen in caspase-9, together with other expected features of the catalytic apparatus, appears in our model. The predicted structure of caspase-9 can serve as a reference for subsite analysis relative to rational design of highly selective caspase inhibitors for therapeutic application.     

Roc, a Ras/GTPase domain in complex proteins

We identified a novel group of the Ras/GTPase superfamily, termed Roc, that is present as domain in complex proteins together with other domains, including leucine-rich repeats (LRRs), ankyrin repeats, WD40 repeats, kinase domains, RasGEF and RhoGAP domains. Roc is always succeeded by a novel 300-400-amino-acid-long domain, termed COR. Proteins with Roc/COR are present in prokaryotes, Dictyostelium, plants and metazoa.     

Female preference for complex/novel signals in a spider

Identifying the various factors that influence complex signalevolution is a difficult task, yet it is fundamental to understandingthe evolution of animal communication. Here we explore the evolutionof complex courtship signaling by taking advantage of a systemin which sexual selection on male courtship traits has driventhe diversification of geographically isolated populations ofthe jumping spider Habronattus pugillis Griswold. Using 2 populations(Santa Rita [SR] and Atascosa [AT]) in which SR females showxenophilic mating preferences for foreign (AT) over local males(SR), we examine the mechanisms driving this preference. BothAT and SR males produce multimodal signals (visual + seismic),and while SR and AT signals share certain seismic components,AT seismic signals are more complex and contain novel components.We conducted mate choice trials where SR females were presentedwith AT or SR males that were either muted or nonmuted. SR femalespreferred to mate and mated more quickly with foreign AT malesover local SR males only if AT males could produce seismic signals(nonmuted treatment). In addition, we found that SR femalesspent a higher proportion of time attentive to foreign AT malesonly if they could produce seismic signals. This evidence suggeststhat SR females have a bias for complex and/or novel forms ofseismic signals.     

Preparation of a highly translocation-competent proOmpA/SecB complex

Methods for reproducibly preparing highly translocation-competent proOmpA were developed. Only a competent form of proOmpA was sorted out from incompetent one using SecB, a translocation-dedicated chaperone, as a probe. Trypsin digestion revealed that the incompetent form of proOmpA was partially folded at its N-terminus, consistent with the jamming of proOmpA within translocon. Although the incompetent form of proOmpA was not active as to topology inversion of SecG, the isolated proOmpA/SecB complex had recovered the ability of SecG inversion. These results let us prepare a proOmpA/SecB complex both in vivo and in vitro that is highly translocation-competent. E. coli cells harboring a plasmid, in which ompA and secB were encoded as a synthetic operon, accumulated the proOmpA/SecB complex in the cytosol. The complex, purified by means of a His tag attached to SecB, was found to be translocation-competent as revealed by the occurrence of SecG inversion, although the signal peptide of proOmpA was sensitive to proteolytic digestion. ProOmpA, in vitro synthesized by means of a continuous exchange cell free system in the presence of SecB-His, was purified as a complex with SecB, which was active as to SecG inversion as well.     

Crystallization of the light-harvesting chlorophyll a/b complex within thylakoid membranes

We have found that treatment of the photosynthetic membranes of green plants, or thylakoids, with the nonionic detergent Triton X-114 at a 10:1 ratio has three effects: (a) photosystem I and coupling factor are solubilized, so that the membranes retain only photosystem II (PS II) and its associated light-harvesting apparatus (LHC-II); (b) LHC-II is crystallized, and so is removed from its normal association with PS II; and (c) LHC-II crystallization causes a characteristic red shift in the 77 degrees K fluorescence from LHC-II. Treatment of thylakoids with the same detergent at a 20:1 ratio results in an equivalent loss of photosystem I and coupling factor, with LHC-II and PS II being retained by the membranes. However, no LHC-II crystals are formed, nor is there a shift in fluorescence. Thus, isolation of a membrane protein is not required for its crystallization, but the conditions of detergent treatment are critical. Membranes with crystallized LHC-II retain tetrameric particles on their surface but have no recognizable stromal fracture face. We have proposed a model to explain these results: LHC-II is normally found within the stromal half of the membrane bilayer and is reoriented during the crystallization process. This reorientation causes the specific fluorescence changes associated with crystallization. Tetrameric particles, which are not changed in any way by the crystallization process, do not consist of LHC-II complexes. PS II appears to be the only other major complex retained by these membranes, which suggests that the tetramers consist of PS II.     

Crystal structure of a beta-catenin/BCL9/Tcf4 complex

The canonical Wnt pathway plays critical roles in embryonic development, stem cell growth, and tumorigenesis. Stimulation of the Wnt pathway leads to the association of beta-catenin with Tcf and BCL9 in the nucleus, resulting in the transactivation of Wnt target genes. We have determined the crystal structure of a beta-catenin/BCL9/Tcf-4 triple complex at 2.6 A resolution. Our studies reveal that the beta-catenin binding site of BCL9 is distinct from that of most other beta-catenin partners and forms a good target for developing drugs that block canonical Wnt/beta-catenin signaling. The BCL9 beta-catenin binding domain (CBD) forms an alpha helix that binds to the first armadillo repeat of beta-catenin, which can be mutated to prevent beta-catenin binding to BCL9 without affecting cadherin or alpha-catenin binding. We also demonstrate that beta-catenin Y142 phosphorylation, which has been proposed to regulate BCL9-2 binding, does not directly affect the interaction of beta-catenin with either BCL9 or BCL9-2.     

Isolation and characterization of a membrane-DNA complex in the mitochondria of Physarum polycephalum

A membrane-DNA complex was isolated by centrifugation of sheared lysate of isolated mitochondria in 20-60% sucrose step solution. Analyses using Hoechst 33258/CsCl density gradient centrifugation and restriction endonuclease treatment showed that DNA in the membrane-DNA complex was AT-rich compared with total mitochondrial DNA (mt DNA) and contained Eco RI fragments of E-4, 5 and 8, which were localized on the right hand of Physarum mitochondrial genome. Phenethyl alcohol (PEA) and ethidium bromide (EB) could disrupt the membrane-DNA complex to release DNA fragments from their complex in vitro. Addition of 0.5% or more PEA, which released 80-90% of the DNA from the membrane-DNA complex in vitro, inhibited not only mitochondrial nuclear division but also mitochondrial division in vivo. EB treatment at more than 1 mg/ml disrupted the membrane-DNA complex in vitro to release 77% of the total DNA in the complex. Addition of 10 micrograms/ml EB induced unequal mitochondrial nuclear division in the microplasmodia, e.g., a dividing dumbbell-shaped mitochondrion had the mt-nucleus in one side and as a result formed then one nucleated and one enucleated mitochondrion. From the EB-pretreated mitochondria, a lesser amount of the membrane-DNA complex was isolated than from the control. These findings mean than the unequal mt-nuclear division is due to dissociation of DNA and the membrane system in the membrane-DNA complex. They strongly suggested that the DNA region (E-4, 5 and 8), where the mitochondrial nucleus is associated with the mitochondrial membrane system plays an important role in mitochondrial nuclear division.     

Chemotaxonomy of the Oscillatoria-Phormidium complex

Five species of Phormidium and eight species of Oscillatoria were compared in terms of carotenoids, biliproteins, protein profiles and α-esterases, leucine aminopeptidase and phosphatase zymograms. The data from protein profiles and α-esterase zymograms support morphological criteria in recognizing four groups of conspecific taxa: O. tenuis, O. amoena, and O. animalis; O. chalybea and O. formosa; P. foveolarum and P. luridum var. olivacea; and P. persicinum and P. ectocarpii. Problems of Cyanophycean taxonomy are considered in terms of biochemical adjuncts to conventional criteria in characterizing and distinguishing species.