Aberrant spindle dynamics and cytokinesis in Dictyostelium discoideum cells that lack glycogen synthase kinase 3

Eukaryotic cell division requires the co-ordinated assembly and disassembly of the mitotic spindle, accurate chromosome segregation and temporal control of cytokinesis to generate two daughter cells. While the absolute details of these processes differ between organisms, there are evolutionarily conserved core components common to all eukaryotic cells, whose identification will reveal the key processes that control cell division. Glycogen synthase kinase 3 (GSK-3) is a major protein kinase found throughout the eukaryotes and regulates many processes, including cell differentiation, growth, motility and apoptosis. In animals, GSK-3 associates with mitotic spindles and its inhibition causes mis-regulation of chromosome segregation. Two suppressor screens in yeast point to a more general effect of GSK-3 on cell division, however the direct role of GSK-3 in control of mitosis has not been explored outside the animal kingdom. Here we report that the Dictyostelium discoideum GSK-3 orthologue, GskA, associates with the mitotic spindle during cell division, as seen for its mammalian counterparts. Dictyostelium possesses only a single GSK-3 gene that can be deleted to eliminate all GSK-3 activity. We found that gskA-null mutants failed to elongate their mitotic spindle and were unable to divide in shaking culture, but have no chromosome segregation defect. These results suggest further conservation for the role of GSK-3 in the regulation of spindle dynamics during mitosis, but also reveal differences in the mechanisms ensuring accurate chromosome segregation.     

Structural and biochemical characterization of Rv2140c,a phosphatidylethanolamine-binding protein from Mycobacterium tuberculosis

Rv2140c is one of many conserved Mycobacterium tuberculosis proteins for which no molecular function has been identified. We have determined a high-resolution crystal structure of the Rv2140c gene product, which reveals a dimeric complex that shares strong structural homology with the phosphatidylethanolamine-binding family of proteins. Rv2140c forms low-millimolar interactions with a selection of soluble phosphatidylethanolamine analogs, indicating that it has a role in lipid metabolism. Furthermore, the small molecule locostatin binds to the Rv2140c ligand-binding site and also inhibits the growth of the model organism Mycobacterium smegmatis.     

Structure and Topology of the Huntingtin 1–17 Membrane Anchor by a Combined Solution and Solid-State NMR Approach

The very amino-terminal domain of the huntingtin protein is directly located upstream of the protein’s polyglutamine tract, plays a decisive role in several important properties of this large protein and in the development of Huntington’s disease. This huntingtin 1–17 domain is on the one hand known to markedly increase polyglutamine aggregation rates and on the other hand has been shown to be involved in cellular membrane interactions. Here, we determined the high-resolution structure of huntingtin 1–17 in dodecyl phosphocholine micelles and the topology of its helical domain in oriented phosphatidylcholine bilayers. Using two-dimensional solution NMR spectroscopy the low-energy conformations of the polypeptide were identified in the presence of dodecyl phosphocholine detergent micelles. In a next step a set of four solid-state NMR angular restraints was obtained from huntingtin 1–17 labeled with ¹⁵N and ²H at selected sites. Of the micellar ensemble of helical conformations only a limited set agrees in quantitative detail with the solid-state angular restraints of huntingtin 1–17 obtained in supported planar lipid bilayers. Thereby, the solid-state NMR data were used to further refine the domain structure in phospholipid bilayers. At the same time its membrane topology was determined and different motional regimes of this membrane-associated domain were explored. The pronounced structural transitions of huntingtin 1–17 upon membrane-association result in a α-helical conformation from K6 to F17, i.e., up to the very start of the polyglutamine tract. This amphipathic helix is aligned nearly parallel to the membrane surface (tilt angle ∼77°) and is characterized by a hydrophobic ridge on one side and an alternation of cationic and anionic residues that run along the hydrophilic face of the helix. This arrangement facilitates electrostatic interactions between huntingtin 1–17 domains and possibly with the proximal polyglutamine tract.     

The Properties of Chondrocyte Membrane Reservoirs and Their Role in Impact-Induced Cell Death

Impact loading of articular cartilage causes extensive chondrocyte death. Cell membranes have a limited elastic range of 3–4% strain but are protected from direct stretch during physiological loading by their membrane reservoir, an intricate pattern of membrane folds. Using a finite-element model, we suggested previously that access to the membrane reservoir is strain-rate-dependent and that during impact loading, the accessible membrane reservoir is drastically decreased, so that strains applied to chondrocytes are directly transferred to cell membranes, which fail when strains exceed 3–4%. However, experimental support for this proposal is lacking. The purpose of this study was to measure the accessible membrane reservoir size for different membrane strain rates using membrane tethering techniques with atomic force microscopy. We conducted atomic force spectroscopy on isolated chondrocytes (n = 87). A micron-sized cantilever was used to extract membrane tethers from cell surfaces at constant pulling rates. Membrane tethers could be identified as force plateaus in the resulting force-displacement curves. Six pulling rates were tested (1, 5, 10, 20, 40, and 80 μm/s). The size of the membrane reservoir, represented by the membrane tether surface areas, decreased exponentially with increasing pulling rates. The current results support our theoretical findings that chondrocytes exposed to impact loading die because of membrane ruptures caused by high tensile membrane strain rates.     

Negative Effects of Conspecific Floral Density on Fruit Set of Two Neotropical Understory Plants

Plant reproductive success is usually positively related to conspecific floral density, but neutral or negative effects of floral density on reproduction have also been reported. Differences in the relationship between reproduction and floral density largely originate from a trade‐off between increasing attractiveness versus increasing competition for pollinators at high floral densities. Although floral densities strongly vary in the understory of tropical forests, for instance, due to variation in light availability, little is known about the density dependence of reproduction in tropical understory plants. We used path analyses to disentangle direct and indirect effects of canopy openness and floral density on fruit set and analyzed the relationship between pollen load and floral density for two Neotropical understory plants, Heliconia metallica and Besleria melancholica. In both species, fruit set was not directly related to canopy openness, but decreased with increasing floral density. In H. metallica, canopy openness had an indirect negative effect on reproduction mediated by its effects on floral density. Effects of floral density on pollen loads were species‐specific. In B. melancholica, pollen loads linearly decreased with increasing floral density, indicating competition for pollinators at high densities. In H. metallica, pollen loads were reduced at both low and high densities, indicating an interplay of facilitative and competitive effects of floral density on pollen deposition. In contrast to other studies, we found negative density dependence of reproduction in both understory species. Negative effects of floral density on reproduction appear to be related to pollinator‐mediated effects on reproduction rather than to variation in abiotic conditions.     

GENETIC DIFFERENTIATION AND SELECTION AGAINST MIGRANTS IN EVOLUTIONARILY REPLICATED EXTREME ENVIRONMENTS

We investigated mechanisms of reproductive isolation in livebearing fishes (genus Poecilia) inhabiting sulfidic and nonsulfidic habitats in three replicate river drainages. Although sulfide spring fish convergently evolved divergent phenotypes, it was unclear if mechanisms of reproductive isolation also evolved convergently. Using microsatellites, we found strongly reduced gene flow between adjacent populations from different habitat types, suggesting that local adaptation to sulfidic habitats repeatedly caused the emergence of reproductive isolation. Reciprocal translocation experiments indicate strong selection against immigrants into sulfidic waters, but also variation among drainages in the strength of selection against immigrants into nonsulfidic waters. Mate choice experiments revealed the evolution of assortative mating preferences in females from nonsulfidic but not from sulfidic habitats. The inferred strength of sexual selection against immigrants (RI_s) was negatively correlated with the strength of natural selection (RI_m), a pattern that could be attributed to reinforcement, whereby natural selection strengthens behavioral isolation due to reduced hybrid fitness. Overall, reproductive isolation and genetic differentiation appear to be replicated and direct consequences of local adaptation to sulfide spring environments, but the relative contributions of different mechanisms of reproductive isolation vary across these evolutionarily independent replicates, highlighting both convergent and nonconvergent evolutionary trajectories of populations in each drainage.     

Genital morphology of female goblin spiders (Arachnida: Araneae: Oonopidae) with functional implications

Spider genital morphology usually provides the best characters for taxonomy. Furthermore, functional genital morphology helps to understand the evolution of complex genitalia and their role in the context of sexual selection. The genital systems of most haplogyne spider families are poorly investigated with respect to their morphology. The present study investigates the female genitalia of the oonopids Oonops pulcher, Oonopinus kilikus, and Pseudotriaeris sp. by means of light microscopy and SEM. The male palps are briefly described. Females of O. pulcher store spermatozoa in an anterior and a posterior receptaculum (PRe). The genitalia resemble the primitive dysderoid genitalia supporting the hypothesis that the subfamily Oonopinae contains more basal oonopids. In O. kilikus, the anterior receptaculum is reduced to a sclerite. Spermatozoa are stored in a PRe. The receptacula of Pseudotriaeris sp. are reduced to sclerites. Spermatozoa in the uterus internus indicate that fertilization happens there or in the ovary. The anterior sclerite might serve females to lock the uterus during copulation as suggested for other gamasomorphines. The male palp of O. kilikus is simple, whereas the palps of O. pulcher and Pseudotriaeris sp. appear more complex. Complicated structures on the palp of Pseudotriaeris sp. indicate that males exert copulatory courtship.     

FAM111A Mutations Result in Hypoparathyroidism and Impaired Skeletal Development

Kenny-Caffey syndrome (KCS) and the similar but more severe osteocraniostenosis (OCS) are genetic conditions characterized by impaired skeletal development with small and dense bones, short stature, and primary hypoparathyroidism with hypocalcemia. We studied five individuals with KCS and five with OCS and found that all of them had heterozygous mutations in FAM111A. One mutation was identified in four unrelated individuals with KCS, and another one was identified in two unrelated individuals with OCS; all occurred de novo. Thus, OCS and KCS are allelic disorders of different severity. FAM111A codes for a 611 amino acid protein with homology to trypsin-like peptidases. Although FAM111A has been found to bind to the large T-antigen of SV40 and restrict viral replication, its native function is unknown. Molecular modeling of FAM111A shows that residues affected by KCS and OCS mutations do not map close to the active site but are clustered on a segment of the protein and are at, or close to, its outer surface, suggesting that the pathogenesis involves the interaction with as yet unidentified partner proteins rather than impaired catalysis. FAM111A appears to be crucial to a pathway that governs parathyroid hormone production, calcium homeostasis, and skeletal development and growth.