The apical annuli of Toxoplasma gondii are composed of coiled‐coil and signalling proteins embedded in the inner membrane complex sutures

The apical annuli are among the most intriguing and understudied structures in the cytoskeleton of the apicomplexan parasite Toxoplasma gondii. We mapped the proteome of the annuli in Toxoplasma by reciprocal proximity biotinylation (BioID), and validated five apical annuli proteins (AAP1–5), Centrin2, and an apical annuli methyltransferase. Moreover, inner membrane complex (IMC) suture proteins connecting the alveolar vesicles were also detected and support annuli residence within the sutures. Super‐resolution microscopy identified a concentric organisation comprising four rings with diameters ranging from 200 to 400 nm. The high prevalence of domain signatures shared with centrosomal proteins in the AAPs together with Centrin2 suggests that the annuli are related and/or derived from the centrosomes. Phylogenetic analysis revealed that the AAPs are conserved narrowly in coccidian, apicomplexan parasites that multiply by an internal budding mechanism. This suggests a role in replication, for example, to provide pores in the mother IMC permitting exchange of building blocks and waste products. However, presence of multiple signalling domains and proteins are suggestive of additional functions. Knockout of AAP4, the most conserved compound forming the largest ring‐like structure, modestly decreased parasite fitness in vitro but had no significant impact on acute virulence in vivo. In conclusion, the apical annuli are composed of coiled‐coil and signalling proteins assembled in a pore‐like structure crossing the IMC barrier maintained during internal budding.     

1H, 13C,and 15N chemical shift assignments of the phosphotyrosine binding domain 2 (PTB2) of human FE65

Phosphotyrosine binding domains (PTB) are protein–protein interaction domains that play important roles in various cellular signal transduction pathways. The second phosphotyrosine binding domain (PTB2) of the human scaffolding protein FE65 interacts with the C-terminal part of the Amyloid Precursor Protein (APP) involved in Alzheimer’s disease. The structure of PTB2 in complex with a 32 amino acid fragment of APP has been solved previously by X-ray crystallography. Here, we report the NMR spectral assignments of the free FE65 PTB2. This provides the basis for further investigation of the interactions of PTB2 with peptides and small organic ligands with the aim of disrupting the PTB2-APP interaction.     

Targeting, Capture, and Stabilization of Microtubules at Early Focal Adhesions

By co-injecting fluorescent tubulin and vinculin into fish fibroblasts we have revealed a “cross talk” between microtubules and early sites of substrate contact. This mutuality was first indicated by the targeting of vinculin-rich foci by microtubules during their growth towards the cell periphery. In addition to passing directly over contact sites, the ends of single microtubules could be observed to target several contacts in succession or the same contact repetitively, with intermittent withdrawals. Targeting sometimes involved side-stepping, or the major re-routing of a microtubule, indicative of a guided, rather than a random process. The paths that microtubules followed into contacts were unrelated to the orientation of stress fiber assemblies and targeting occurred also in mouse fibroblasts that lacked a system of intermediate filaments. Further experiments with microtubule inhibitors showed that adhesion foci can: (a) capture microtubules and stabilize them against disassembly by nocodazole; and (b), act as preferred sites of microtubule polymerization, during either early recovery from nocodazole, or brief treatment with taxol. From these and other findings we speculate that microtubules are guided into substrate contact sites and through the motor-dependent delivery of signaling molecules serve to modulate their development. It is further proposed this modulation provides the route whereby microtubules exert their influence on cell shape and polarity.     

Characterization of soluble vs membrane-bound human placental 5'-nucleotidase

Three forms of 5'-nucleotidase purified from human placenta (two membrane-bound forms, one sensitive and one resistant to cleavage by phosphatidylinositol-specific phospholipase C, as well as a soluble form) had the same molecular weight before (73,000 Da) and after (56,000 Da) digestion with N-glycosidase F and showed similar amino acid compositions, N-terminal amino acid sequences, and KMs for IMP (9.6 to 11.9 microM). Thus, these three forms of 5'-nucleotidase appear to have very similar structures. The form sensitive to phosphatidylinositol-specific phospholipase C contained nearly 1 mol myo-inositol/mol of protein as determined by mass spectrometry, indicating a glycosyl phosphatidylinositol membrane anchor. Soluble 5'-nucleotidase contained a similar quantity of myo-inositol, suggesting that it was previously membrane-anchored via glycosyl phosphatidylinositol. The form resistant to phosphatidylinositol-specific phospholipase C contained less myo-inositol, leaving open the possibility of a third form of 5'-nucleotidase with a conventional transmembrane anchor.     

Moulting starvation in emperor penguin (Aptenodytes forsten) chicks

Summary The moult fast in emperor penguin (Aptenodytes forsteri) chicks was studied during January 1990 at Drescher Inlet, eastern Weddell Sea. In early January feeding of the chicks had stopped and about 4,000–5,000 chicks were in the inlet. The number of starving chicks decreased rapidly until 26 January when all chicks had either left the inlet or died. Mean body mass loss of starving chicks was 257 g/day and the evaluated specific daily mass loss was 25 g/kg body mass. The critical body mass, i.e. the mass below which chicks die, during moulting starvation was estimated to be 4 kg. Mean body mass was higher and mass loss lower in chicks at more advanced moult stages. Chicks left the inlet before moult was completed, although the sea-ice was still stable.     

Southern rockhopper penguin Eudyptes chrysocome chrysocome foraging at Possession Island

The foraging ecology of rockhopper penguins was studied at Possession Island, southern Indian Ocean, by counting the number of birds departing from and arriving at colonies over the course of the day and by equipping three birds with time/depth loggers, one of which was recovered having recorded a total of 12 days foraging activity. Both the counts and the results from the diving behaviour showed that the birds foraged exclusively diurnally. Maximum dive depth was 66 m although most time was spent between 10 and 25 m, depths that did not accord with the published distribution of their principal prey as detected by nets and acoustics. Received: 29 March 1996/Accepted: 10 June 1996     

Regulation of S-layer protein synthesis of Bacillus stearothermophilus PV72 through variation of continuous cultivation conditions

The crystalline cell surface layer (S-layer) of Bacillus stearothermophilus PV72 shows hexagonal lattice symmetry and is composed of a single protein species with a molecular weight of 130000. For investigating the regulation of S-layer protein synthesis, Bacillus stearothermophilus PV72 was grown in continuous culture on synthetic PVIII- medium with glucose as carbon source at constant dilution rate of 0.3 h⁻¹ at 57 ° C under different conditions and limitations. A complete outer S-layer and an S-layer protein pool sufficient for formation of about 70% inner S-layer was produced under carbon-limited growth. The inner S-layer results from an S-layer protein pool stored in the peptidoglycan-containing layer of whole cells which can emerge and assemble on the inner face of the rigid cell wall layer during the cell wall preparation procedure. Under oxygen-limited growth, only a complete outer S-layer but no S-layer protein pool was synthesized. Reduction of the methionine concentration of PVIII-medium from 100 to 10 mg l⁻¹ led to a clear decrease in S-layer protein production and to an incomplete outer S-layer. During growth in the presence of excess glucose, S-layer protein synthesis was replaced by that of an exopolysaccharide matrix. After changing to carbon limitation again, the original level of S-layer protein synthesis was achieved after only four volume exchanges. Feeding of casein hydrolysate or aromatic or basic amino acids to the continuous culture induced an irreversible loss of S-layer protein synthesis after from five to ten volume exchanges. In contrast, addition of Gly, Ala, Val, Leu, Ile, Glu, Gln, Asp, Asn, Ser and Thr in different mixtures could significantly stimulate S-layer protein production.