PRC1‐labeled microtubule bundles and kinetochore pairs show one‐to‐one association in metaphase

In the mitotic spindle, kinetochore microtubules form k‐fibers, whereas overlap or interpolar microtubules form antiparallel arrays containing the cross‐linker protein regulator of cytokinesis 1 (PRC1). We have recently shown that an overlap bundle, termed bridging fiber, links outermost sister k‐fibers. However, the relationship between overlap bundles and k‐fibers throughout the spindle remained unknown. Here, we show that in a metaphase spindle more than 90% of overlap bundles act as a bridge between sister k‐fibers. We found that the number of PRC1‐GFP‐labeled bundles per spindle is nearly the same as the number of kinetochore pairs. Live‐cell imaging revealed that kinetochore movement in the equatorial plane of the spindle is highly correlated with the movement of the coupled PRC1‐GFP‐labeled fiber, whereas the correlation with other fibers decreases with increasing distance. Analysis of endogenous PRC1 localization confirmed the results obtained with PRC1‐GFP. PRC1 knockdown reduced the bridging fiber thickness and interkinetochore distance throughout the spindle, suggesting a function of PRC1 in bridging microtubule organization and force balance in the metaphase spindle

Seril-tRNA-sintetaze iz metanogenih arheja: supresija bakterijskih amber mutacija i heterologna toksičnost

Methanogenic archaea possess unusual seryl-tRNA synthetases (SerRS), evolutionarily distinct from the SerRSs found in other archaea, eucaryotes and bacteria. Our recent X-ray structural analysis of Methanosarcina barkeri SerRS revealed an idiosyncratic N-terminal domain and catalytic zinc ion in the active site. To shed further light on substrate discrimination by methanogenic-type SerRS, we set up to explore in vivo the interaction of methanogenic-type SerRSs with their cognate tRNAs in Escherichia coli or Saccharomyces cerevisiae. The expression of various methanogenic-type SerRSs was toxic for E. coli, resulting in the synthesis of erroneous proteins, as revealed by β-galactosidase stability assay. Although SerRSs from methanogenic archaea recognize tRNAsSer from all three domains of life in vitro, the toxicity presumably precluded the complementation of endogenous SerRS function in both, E. coli and S. cerevisiae. However, despite the observed toxicity, coexpression of methanogenic-type SerRS with its cognate tRNA suppressed bacterial amber mutation.Metanogene arheje imaju neobične seril-tRNA-sintetaze (SerRS), evolucijski udaljene od SerRS koje se mogu naći u drugih arheja, eukariota i bakterija. Naša nedavna analiza kristalne strukture SerRS iz metanogene arheje Methanosarina barkeri otkrila je karakterističnu N-terminalnu domenu i katalitički ion cinka na aktivnom mjestu. Da bi se rasvijetlio način na koji metanogeni tip SerRS diskriminira supstrate, autori su istraživali in vivo interakciju metanogenog tipa SerRS s pripadnim molekulama tRNA u bakteriji Escherichia coli ili kvascu Saccharomyces cerevisiae. Ekspresija raznih SerRS metanogenoga tipa bila je toksična za bakteriju E. coli, te je rezultirala sintezom proteina s greškama u aminokiselinskom sastavu, što se vidi iz testa stabilnosti β-galaktozidaze. Iako je SerRS iz metanogenih arheja mogla prepoznati tRNASer iz sve tri domene života in vitro, vjerojatno je toksičnost zasjenila komplementaciju funkcije endogene SerRS u bakteriji E. coli i kvascu S. cerevisiae. Međutim, unatoč toksičnosti, koekspresija metanogenoga tipa SerRS s pripadnom tRNA suprimirala je bakterijsku amber mutaciju

Seryl-tRNA Synthetases from Methanogenic Archaea: Suppression of Bacterial Amber Mutation and Heterologous Toxicity

Methanogenic archaea possess unusual seryl-tRNA synthetases (SerRS), evolutionarily distinct from the SerRSs found in other archaea, eucaryotes and bacteria. Our recent X-ray structural analysis of Methanosarcina barkeri SerRS revealed an idiosyncratic N-terminal domain and catalytic zinc ion in the active site. To shed further light on substrate discrimination by methanogenic-type SerRS, we set up to explore in vivo the interaction of methanogenic-type SerRSs with their cognate tRNAs in Escherichia coli or Saccharomyces cerevisiae. The expression of various methanogenic-type SerRSs was toxic for E. coli, resulting in the synthesis of erroneous proteins, as revealed by β-galactosidase stability assay. Although SerRSs from methanogenic archaea recognize tRNAsSer from all three domains of life in vitro, the toxicity presumably precluded the complementation of endogenous SerRS function in both, E. coli and S. cerevisiae. However, despite the observed toxicity, coexpression of methanogenic-type SerRS with its cognate tRNA suppressed bacterial amber mutation

LIPG-promoted lipid storage mediates adaptation to oxidative stress in breast cancer

Endothelial lipase (LIPG) is a cell surface associated lipase that displays phospholipase A1 activity towards phosphatidylcholine present in high‐density lipoproteins (HDL). LIPG was recently reported to be expressed in breast cancer and to support proliferation, tumourigenicity and metastasis. Here we show that severe oxidative stress leading to AMPK activation triggers LIPG upregulation, resulting in intracellular lipid droplet accumulation in breast cancer cells, which supports survival. Neutralizing oxidative stress abrogated LIPG upregulation and the concomitant lipid storage. In human breast cancer, high LIPG expression was observed in a limited subset of tumours and was significantly associated with shorter metastasis‐free survival in node‐negative, untreated patients. Moreover, expression of PLIN2 and TXNRD1 in these tumours indicated a link to lipid storage and oxidative stress. Altogether, our findings reveal a previously unrecognized role for LIPG in enabling oxidative stress‐induced lipid droplet accumulation in tumour cells that protects against oxidative stress, and thus supports tumour progression

Identification of Amino Acids in the N-terminal Domain of Atypical Methanogenic-type Seryl-tRNA Synthetase Critical for tRNA Recognition*

Seryl-tRNA synthetase (SerRS) from methanogenic archaeon Methanosarcina barkeri, contains an idiosyncratic N-terminal domain, composed of an antiparallel β-sheet capped by a helical bundle, connected to the catalytic core by a short linker peptide. It is very different from the coiled-coil tRNA binding domain in bacterial-type SerRS. Because the crystal structure of the methanogenic-type SerRS·tRNA complex has not been obtained, a docking model was produced, which indicated that highly conserved helices H2 and H3 of the N-terminal domain may be important for recognition of the extra arm of tRNASer. Based on structural information and the docking model, we have mutated various positions within the N-terminal region and probed their involvement in tRNA binding and serylation. Total loss of activity and inability of the R76A variant to form the complex with cognate tRNA identifies Arg76 located in helix H2 as a crucial tRNA-interacting residue. Alteration of Lys79 positioned in helix H2 and Arg94 in the loop between helix H2 and β-strand A4 have a pronounced effect on SerRS·tRNASer complex formation and dissociation constants (KD) determined by surface plasmon resonance. The replacement of residues Arg38 (located in the loop between helix H1 and β-strand A2), Lys141 and Asn142 (from H3), and Arg143 (between H3 and H4) moderately affect both the serylation activity and the KD values. Furthermore, we have obtained a striking correlation between these results and in vivo effects of these mutations by quantifying the efficiency of suppression of bacterial amber mutations, after coexpression of the genes for M. barkeri suppressor tRNASer and a set of mMbSerRS variants in Escherichia coli