Use of the novel Plk1 inhibitor ZK-thiazolidinone to elucidate functions of Plk1 in early and late stages of mitosis

Polo-like kinase 1 (Plk1) is a key regulator of mitotic progression and cell division in eukaryotes. It is highly expressed in tumor cells and considered a potential target for cancer therapy. Here, we report the discovery and application of a novel potent small-molecule inhibitor of mammalian Plk1, ZK-Thiazolidinone (TAL). We have extensively characterized TAL in vitro and addressed TAL specificity within cells by studying Plk1 functions in sister chromatid separation, centrosome maturation, and spindle assembly. Moreover, we have used TAL for a detailed analysis of Plk1 in relation to PICH and PRC1, two prominent interaction partners implicated in spindle assembly checkpoint function and cytokinesis, respectively. Specifically, we show that Plk1, when inactivated by TAL, spreads over the arms of chromosomes, resembling the localization of its binding partner PICH, and that both proteins are mutually dependent on each other for correct localization. Finally, we show that Plk1 activity is essential for cleavage furrow formation and ingression, leading to successful cytokinesis.     

Mechanical strain stimulates COPII‐dependent secretory trafficking via Rac1

Cells are constantly exposed to various chemical and physical stimuli. While much has been learned about the biochemical factors that regulate secretory trafficking from the endoplasmic reticulum (ER), much less is known about whether and how this trafficking is subject to regulation by mechanical signals. Here, we show that subjecting cells to mechanical strain both induces the formation of ER exit sites (ERES) and accelerates ER‐to‐Golgi trafficking. We found that cells with impaired ERES function were less capable of expanding their surface area when placed under mechanical stress and were more prone to develop plasma membrane defects when subjected to stretching. Thus, coupling of ERES function to mechanotransduction appears to confer resistance of cells to mechanical stress. Furthermore, we show that the coupling of mechanotransduction to ERES formation was mediated via a previously unappreciated ER‐localized pool of the small GTPase Rac1. Mechanistically, we show that Rac1 interacts with the small GTPase Sar1 to drive budding of COPII carriers and stimulates ER‐to‐Golgi transport. This interaction therefore represents an unprecedented link between mechanical strain and export from the ER.     

Identity and change: The example of the falashas,between assimilation in Ethiopia and integration in Israel

Daniel Friedmann is a Research Fellow at the C.N.R.S., Paris. Ulysses Santamaria is a Fellow at the Maison des Sciences de L'Homme, Paris.     

Polyhomeotic: A gene of Drosophila melanogaster required for correct expression of segmental identity

Summary A new locus in Drosophila melanogaster that is required for the correct expression of segmental identity has been discovered. The new locus, termed polyhomeotic (ph), is X-linked and maps cytologically to bands 2D2-3. Homozygous ph flies have homeotic transformations similar to those of known dominant gain of function mutants in the Antennapedia and bithorax complexes (ANT-C, BX-C), and in addition show loss of the humerus. ph interacts with three other similar mutations: Polycomb (Pc), Polycomblike (Pcl), and extra sex comb (esc), and acts as a dominant enhancer of Pc. The expression of ph depends on the ANT-C and BX-C dosage. ph has no embryonic phenotype, but temperature shift studies on ph ² show that the ph ⁺ product is required during embryogenesis and larval development. We propose that ph mutants in some way disrupt the normal expression of the ANT-C and BX-C, and, therefore, that ph ⁺ is needed for maintenance of segmental identity.     

The lack of control of water loss in micropropagated plants is not related to poor cuticle development

To assess if cuticular transpiration could contribute significantly to the high rates of water loss often observed in micropropagated plants after transfer to the nursery, it was tested whether adaxial cuticular water permeance (P) of leaves grown in vitro was higher than that of leaves grown ex vitro. For four species of micropropagated plants with hypostous leaves ( Delphinium elatum hybrid, Doronicum hybrid, Hosta sieboldiana var. elegans, Rodgersia pinnata ), P was determined with two independent techniques which gave similar results. Minimum adaxial overall conductance was measured with the same methods for a Heuchera hybrid which had amphistomatous leaves. Leaves of all species except Heuchera lost 36–65% of their original weight within 25 min after excision. Detached leaves whose abaxial surfaces had been coated lost only 25–38% of their original weight within 5–9 h. Permeances (P) were between 1 × 10⁻⁵ m s⁻¹ and 1 × 10⁻⁴ m s⁻¹, which was within the range of typical values found with leaves grown ex vitro. From these results and a critical assessment of the literature it is concluded that there is no evidence that P of micropropagated plants was high enough to contribute significantly to the desiccation problem at the transfer stage.     

Folding of the polypeptide chain(s), conformational flexibility and reactivity of the metal active site of hemocyanin and tyrosinase

Summary The comparative accessibility of the active sites of hemocyanin and tyrosinase, two proteins containing a binuclear type-3 copper site, has been investigated. The approaches were: (a) the kinetic study of the reaction of hemocyanin with cyanide in the presence of conformation perturbants; (b) the comparison between the kinetic parameters of the cyanide reaction on hemocyanin and tyrosinase; (c) the study of the efficiency and reaction mechanism of hemocyanin interaction with a typical tyrosinase substrate like catechol. The results indicate that the active site of tyrosinase is much more exposed than that of hemocyanin.     

Interactions Crucial for Three-Dimensional Domain Swapping in the HP-RNase Variant PM8

The structural determinants that are responsible for the formation of higher order associations of folded proteins remain unknown. We have investigated the role on the dimerization process of different residues of a domain-swapped dimer human pancreatic ribonuclease variant. This variant is a good model to study the dimerization and swapping processes because dimer and monomer forms interconvert, are easily isolated, and only one dimeric species is produced. Thus, simple models for the swapping process can be proposed. The dimerization (dissociation constant) and swapping propensity have been studied using different variants with changes in residues that belong to different putative molecular determinants of dimerization. Using NMR spectroscopy, we show that these mutations do not substantially alter the overall conformation and flexibility, but affect the residue level stability. Overall, the most critical residues for the swapping process are those of one subunit that interact with the hinge loop of another one-subunit residue, stabilizing it in a conformation that favors the interchange. Tyr²⁵, Gln¹⁰¹, and Pro¹⁹, with Asn¹⁷, Ser²¹, and Ser²³, are found to be the most significant; notably, Glu¹⁰³ and Arg¹⁰⁴, which were postulated to form salt bridges that would stabilize the dimer, are not critical for dimerization.