Role of the Kinesin-like Protein KipB in Aspergillus nidulans

Molecular motors are protein machines, which power almost all forms of movement in the living world. Among the best known are the motors that hydrolyze ATP and use the derived energy to generate force. They are involved in a variety of diverse cellular functions as vesicle and organelle transport, cytoskeleton dynamics, morphogenesis, polarized growth, cell movements, spindle formation, chromosome movement, nuclear fusion, and signal transduction. Three superfamilies of molecular motors, kinesins, dyneins, and myosins, have so far been well characterized. These motors use microtubules (in the case of kinesines and dyneins) or actin filaments (in the case of myosins) as tracks to transport cargo materials within a cell. Analysis of fungal genomes revealed at least 10 distinct kinesins in filamentous fungi, some of which are not found in yeasts. We used the motor domain of conventional kinesin (KinA) from Aspergillus nidulans to perfom BLAST searches at the public A. nidulans genome database, at the Whitehead Center for Genome Research (Cambridge USA), and identified eleven putative kinesin motors. They grouped into nine of the eleven families, two kinesins being found in the Unc104 familiy and interestingly, one did not fall into any of the known families. The present work analyses the function of a kinesin-like protein in A. nidulans, KipB, which is a member of the Kip3 kinesin family. This family includes one representative in Saccharomyces cerevisiae (Kip3, the family founding member), two in Schizosaccharomyces pombe, Klp5 and Klp6 and one in Drosophila, Klp67A, the single one reported so far for higher eukaryotes in this family. Kip3 kinesins are implicated in microtubule disassembly and are required for chromosome segregation in mitosis and meiosis. To assess the function of KipB kinesin in A. nidulans, a kipB disruption strain was constructed. Analysis of the DkipB mutant revealed new features concerning the cellular functions of Kip3 proteins, but also some conserved ones. kipB is not essential for vegetative growth, and meiosis and ascospore formation were not affected in the DkipB mutant. The KipB protein was shown to be involved in the turnover of interphase cytoplasmic, mitotic and astral microtubules. DkipB mutants are less sensitive to the microtubule-destabilizing drug benomyl, and the microtubule cytoskeleton of interphase cells in DkipB mutants appears altered. Interestingly, spindle morphology and positioning were severely affected. Spindles were highly mobile, could overpass each other, moved over long distances through the cytoplasm, and displayed in 64% of the cases an extremely bent shape, latter feature being the first time reported for Kip3 kinesins. Mitotic progression was delayed in the DkipB mutant and a higher number of cytoplasmic microtubules remained intact during mitosis. DkipB heterozygous strains showed an increased instability of diploid nuclei, which proved once more KipB involvement in mitosis, along with DkipB clear genetic interaction with a mutation in another mitotic kinesin in A. nidulans, bimC4. An N-terminal GFP-KipB construct localized to cytoplasmic microtubules in interphase cells and to spindle and astral microtubules during mitosis, in a discontinuous pattern. Speckles of GFP-KipB appeared to be aligned in the cell. Time-lapse video microscopy indicated that the spots were moving independently towards the microtubule plus ends. This advanced the hypothesis that KipB could display processivity and intrinsic motility along microtubules, or that other kinesins involved in organelle motility are able to target the KipB protein to the microtubule plus ends. In the case of C-terminally truncated GFP-KipB protein versions, a stronger GFP signal was obtained and colocalization with a-tubulin-GFP revealed that they uniformly stain cytoplasmic, mitotic and astral microtubules. This suggests that the C-terminus is important for the correct localization and the movement of KipB protein along microtubules

Report on the Verification of the Performance of GHB614 and LLCotton25 Event-specific PCR-based Methods Applied to DNA Extracted from Stack Cotton GHB614 x LLCotton25

An application was submitted by Bayer CropScience AG to request the authorisation of genetically modified cotton stack GHB614 x LLCotton25 (tolerant to glufosinate ammonium and glyphosate-containing herbicides) and all sub-combinations of the individual events as present in the segregating progeny, for food and feed uses, and import and processing, in accordance with articles 5 and 17 of Regulation (EC) N° 1829/2003 GM Food and GM Feed. The unique identifier assigned to GHB614 x LLCotton25 cotton is BCS-GHØØ2-5xACS-GHØØ1-3. The genetically modified cotton stack GHB614 x LLCotton25 has been obtained by conventional crossing of two genetically modified cotton events: GHB614 and LLCotton25, without any new genetic modification. The EU-RL GMFF has previously validated individually, and declared fit for purpose, the detection methods for the single events GHB614 and LLCotton25 (see http://gmo-crl.jrc.ec.europa.eu/StatusOfDossiers.aspx). In line with the approach defined by the ENGL (http://gmo-crl.jrc.ec.europa.eu/doc/Min_ Perf_Requirements_Analytical_methods.pdf) the EU-RL GMFF has carried out only an in-house verification of the performance of each validated method when applied to DNA extracted from GHB614 x LLCotton25. The herewith reported in-house verification study lead to the conclusion that the individual methods meet the ENGL performance criteria also when applied to DNA extracted from the GM cotton stack GHB614 x LLCotton25.JRC.I.3 - Molecular Biology and Genomic

Transcription factor Sp3 as target for SUMOylation in vivo

A group of sequence-specific DNA-binding proteins related to the transcription factor Sp1 (specificity protein 1) has been implicated in the regulation of many different genes, since binding sites for these transcription factors (GC/GT boxes) are a recurrent motif in regulatory sequences of these genes. In contrast to the transcriptional activators Sp1 and Sp4, the ubiquitously expressed Sp3 protein can both activate and repress transcription. The complex activity of Sp3 depends on two glutamine-rich activation domains, similar to those found in Sp1 and Sp4, and, adjacent to these, on an inhibitory domain unique to Sp3. The critical lysine residue in the Sp3 inhibitory domain lies within a consensus motif (IK551EE) that targets proteins for SUMO modification. SUMO (small ubiquitin-related modifier) is covalently attached to lysine residues in target proteins via an isopeptide linkage in a multi-step process that is analogous to ubiquitination. The present work analyses various aspects of SUMO conjugation to Sp3 in vivo. Studying modification of Sp3 by SUMO is complicated by the existence of a number of Sp3 isoforms. Immunoblot analyses revealed four distinct Sp3 proteins, two slow migrating of more than 100 kDa and two fast migrating species. Seven to eight Sp3 bands appeared, when cells were lysed in denaturing conditions. The additional protein species represent SUMO modified Sp3 isoforms. Currently, it is not known whether the relative distribution of the different Sp3 isoforms is regulated. However, a significant shift towards the long isoforms of Sp3, however, is observed in Sp1-/- ES cells demonstrating that Sp3 isoform expression principally can change in vivo. In addition, this observation suggests that the long isoforms of Sp3 may take over Sp1 functions under Sp1 knockout conditions. When Sp3 is overexpressed along with SUMO1 and SUMO2 in cells in culture, attachment of both SUMO paralogues to Sp3 occurred with almost equal efficiency. Beside lysine 551 within the inhibitory domain, there are two other potential SUMOylation sites in Sp3 (VKQE at position 9 and IKDE at position 120). This study revealed that SUMOylation takes place exclusively at K551, present in all four isoforms. Visualization of endogenous Sp3 by immumofluorescence showed a sponge-like, diffuse appearance, located predominantly in the nucleus. Evolutionally closely related Sp family members Sp1 and Sp2 are also located in the nucleus and the subcellular localization patterns are similar to Sp3. Ectopic expression of SUMO1 fused to GFP (green fluorescent protein) led to the accumulation of this fusion protein within subnuclear “dots” or PODs (promyelocytic leukemia oncogenic domains), whereas endogenous Sp3 remained diffusely distributed throughout nuclei. In addition, the wild-type Sp3 isoforms and the SUMOylation-deficient mutants of Sp3were located in the nucleus exhibiting also a sponge-like, diffuse appearance. Analyzing the Sp3 expression in different cell lines and mouse organs revealed that the relative level of Sp3 modification by SUMO is not cell line or organ dependent. In addition, no variation in Sp3 expression pattern after serum starvation, serum induction and heat shock was observed. Ultraviolet radiation or Tumor Necrosis Factor alpha and Cycloheximide treatment of mammalian cells did not alter the SUMOylation level of Sp3 protein in our experimental conditions. A significant reduction in Sp3 SUMO modification was observed upon treatment with MG-132, a cell-permeable inhibitor of the proteasome. Possibly this proteasome inhibitor prevents proteasome degradation of SUMO specific isopeptidase, which subsequently remove the Sp3-SUMO moiety. PIAS1 (protein inhibitor of activated STAT) was previously cloned in a two-hybrid screen by using the inhibitory domain of Sp3. Moreover, it was shown that PIAS1 strongly enhances SUMO-modification of Sp3 in vitro and thus acts as an SUMO E3 ligase towards Sp3. PIAS1-associated proteins might confer substrate specificity towards Sp3 and other transcription factors and/or regulate PIAS1 activity in vivo. For the purification and identification of PIAS1-associated proteins, a number of C-terminal tagged expression plasmids were constructed for constitutive and inducible expression. The dual-tag affinity purification system established in this thesis work contains a small 15 amino acid artificial tag (BiotinTAG) that becomes biotinylated by the BirA ligase upon co-transfection of an appropriate expression construct. To enhance specificity, a second tag was included in the expression vectors (Calmodulin Binding Peptide or alternatively FLAG or Triple-FLAG). In addition, dual tags expression plasmids for Sp3 were constructed. The establishment of stable cell lines expressing these fusion proteins in an inducible manner was initiated. Such cell lines might be ideal for further analyzes of PIAS1 activities and to purify PIAS1 (Sp3) associated factors

Report on the Verification of the Performance of Bt11, MIR162, MIR604 and GA21 Event-specific PCR-based Methods Applied to DNA Extracted from Stack Maize Bt11 x MIR162 x MIR604 x GA21

An application was submitted by Syngenta Crop Protection AG to request the authorisation of genetically modified Bt11 x MIR162 x MIR604 x GA21 maize (resistant to lepidopteran and coleopteran pests, able to utilise mannose as the only primary carbon source and tolerant to glufosinate ammonium and glyphosate) and all sub-combinations of the individual events as present in the segregating progeny, for food and feed uses, and import and processing, in accordance with articles 5 and 17 of Regulation (EC) N° 1829/2003 GM Food and GM Feed. The unique identifier assigned to Bt11 x MIR162 x MIR604 x GA21 maize is SYN-BTØ11-1x YN-IR162-4xSYN-IR6Ø4-5xMON-ØØØ21-9. The genetically modified maize line Bt11 x MIR162 x MIR604 x GA21 maize has been obtained by conventional crossing of four genetically modified maize events: Bt11, MIR162, MIR604, and GA21 without any new genetic modification. The EU-RL GMFF has previously validated individually, and declared fit for purpose, the detection methods for the single events Bt11, MIR162, MIR604 and GA21 (see http://gmo-crl.jrc.ec.europa.eu/StatusOfDossiers.aspx). In line with the approach defined by the ENGL (http://gmo-crl.jrc.ec.europa.eu/doc/Min_Perf_Requirements_Analytical_methods.pdf) the EU-RL GMFF therefore has carried out only an in-house verification of the performance of each validated method when applied to DNA extracted from Bt11 x MIR162 x MIR604 x GA21. The hereby reported the in-house verification study lead to the conclusion that the individual methods meet the ENGL criteria also when applied to DNA extracted from the GM maize stack Bt11 x MIR162 x MIR604 x GA21JRC.I.3-Molecular Biology and Genomic

Transcription factor Sp3 as target for SUMOylation in vivo

A group of sequence-specific DNA-binding proteins related to the transcription factor Sp1 (specificity protein 1) has been implicated in the regulation of many different genes, since binding sites for these transcription factors (GC/GT boxes) are a recurrent motif in regulatory sequences of these genes. In contrast to the transcriptional activators Sp1 and Sp4, the ubiquitously expressed Sp3 protein can both activate and repress transcription. The complex activity of Sp3 depends on two glutamine-rich activation domains, similar to those found in Sp1 and Sp4, and, adjacent to these, on an inhibitory domain unique to Sp3. The critical lysine residue in the Sp3 inhibitory domain lies within a consensus motif (IK551EE) that targets proteins for SUMO modification. SUMO (small ubiquitin-related modifier) is covalently attached to lysine residues in target proteins via an isopeptide linkage in a multi-step process that is analogous to ubiquitination. The present work analyses various aspects of SUMO conjugation to Sp3 in vivo. Studying modification of Sp3 by SUMO is complicated by the existence of a number of Sp3 isoforms. Immunoblot analyses revealed four distinct Sp3 proteins, two slow migrating of more than 100 kDa and two fast migrating species. Seven to eight Sp3 bands appeared, when cells were lysed in denaturing conditions. The additional protein species represent SUMO modified Sp3 isoforms. Currently, it is not known whether the relative distribution of the different Sp3 isoforms is regulated. However, a significant shift towards the long isoforms of Sp3, however, is observed in Sp1-/- ES cells demonstrating that Sp3 isoform expression principally can change in vivo. In addition, this observation suggests that the long isoforms of Sp3 may take over Sp1 functions under Sp1 knockout conditions. When Sp3 is overexpressed along with SUMO1 and SUMO2 in cells in culture, attachment of both SUMO paralogues to Sp3 occurred with almost equal efficiency. Beside lysine 551 within the inhibitory domain, there are two other potential SUMOylation sites in Sp3 (VKQE at position 9 and IKDE at position 120). This study revealed that SUMOylation takes place exclusively at K551, present in all four isoforms. Visualization of endogenous Sp3 by immumofluorescence showed a sponge-like, diffuse appearance, located predominantly in the nucleus. Evolutionally closely related Sp family members Sp1 and Sp2 are also located in the nucleus and the subcellular localization patterns are similar to Sp3. Ectopic expression of SUMO1 fused to GFP (green fluorescent protein) led to the accumulation of this fusion protein within subnuclear “dots” or PODs (promyelocytic leukemia oncogenic domains), whereas endogenous Sp3 remained diffusely distributed throughout nuclei. In addition, the wild-type Sp3 isoforms and the SUMOylation-deficient mutants of Sp3were located in the nucleus exhibiting also a sponge-like, diffuse appearance. Analyzing the Sp3 expression in different cell lines and mouse organs revealed that the relative level of Sp3 modification by SUMO is not cell line or organ dependent. In addition, no variation in Sp3 expression pattern after serum starvation, serum induction and heat shock was observed. Ultraviolet radiation or Tumor Necrosis Factor alpha and Cycloheximide treatment of mammalian cells did not alter the SUMOylation level of Sp3 protein in our experimental conditions. A significant reduction in Sp3 SUMO modification was observed upon treatment with MG-132, a cell-permeable inhibitor of the proteasome. Possibly this proteasome inhibitor prevents proteasome degradation of SUMO specific isopeptidase, which subsequently remove the Sp3-SUMO moiety. PIAS1 (protein inhibitor of activated STAT) was previously cloned in a two-hybrid screen by using the inhibitory domain of Sp3. Moreover, it was shown that PIAS1 strongly enhances SUMO-modification of Sp3 in vitro and thus acts as an SUMO E3 ligase towards Sp3. PIAS1-associated proteins might confer substrate specificity towards Sp3 and other transcription factors and/or regulate PIAS1 activity in vivo. For the purification and identification of PIAS1-associated proteins, a number of C-terminal tagged expression plasmids were constructed for constitutive and inducible expression. The dual-tag affinity purification system established in this thesis work contains a small 15 amino acid artificial tag (BiotinTAG) that becomes biotinylated by the BirA ligase upon co-transfection of an appropriate expression construct. To enhance specificity, a second tag was included in the expression vectors (Calmodulin Binding Peptide or alternatively FLAG or Triple-FLAG). In addition, dual tags expression plasmids for Sp3 were constructed. The establishment of stable cell lines expressing these fusion proteins in an inducible manner was initiated. Such cell lines might be ideal for further analyzes of PIAS1 activities and to purify PIAS1 (Sp3) associated factors

Role of the Kinesin-like Protein KipB in Aspergillus nidulans

Molecular motors are protein machines, which power almost all forms of movement in the living world. Among the best known are the motors that hydrolyze ATP and use the derived energy to generate force. They are involved in a variety of diverse cellular functions as vesicle and organelle transport, cytoskeleton dynamics, morphogenesis, polarized growth, cell movements, spindle formation, chromosome movement, nuclear fusion, and signal transduction. Three superfamilies of molecular motors, kinesins, dyneins, and myosins, have so far been well characterized. These motors use microtubules (in the case of kinesines and dyneins) or actin filaments (in the case of myosins) as tracks to transport cargo materials within a cell. Analysis of fungal genomes revealed at least 10 distinct kinesins in filamentous fungi, some of which are not found in yeasts. We used the motor domain of conventional kinesin (KinA) from Aspergillus nidulans to perfom BLAST searches at the public A. nidulans genome database, at the Whitehead Center for Genome Research (Cambridge USA), and identified eleven putative kinesin motors. They grouped into nine of the eleven families, two kinesins being found in the Unc104 familiy and interestingly, one did not fall into any of the known families. The present work analyses the function of a kinesin-like protein in A. nidulans, KipB, which is a member of the Kip3 kinesin family. This family includes one representative in Saccharomyces cerevisiae (Kip3, the family founding member), two in Schizosaccharomyces pombe, Klp5 and Klp6 and one in Drosophila, Klp67A, the single one reported so far for higher eukaryotes in this family. Kip3 kinesins are implicated in microtubule disassembly and are required for chromosome segregation in mitosis and meiosis. To assess the function of KipB kinesin in A. nidulans, a kipB disruption strain was constructed. Analysis of the DkipB mutant revealed new features concerning the cellular functions of Kip3 proteins, but also some conserved ones. kipB is not essential for vegetative growth, and meiosis and ascospore formation were not affected in the DkipB mutant. The KipB protein was shown to be involved in the turnover of interphase cytoplasmic, mitotic and astral microtubules. DkipB mutants are less sensitive to the microtubule-destabilizing drug benomyl, and the microtubule cytoskeleton of interphase cells in DkipB mutants appears altered. Interestingly, spindle morphology and positioning were severely affected. Spindles were highly mobile, could overpass each other, moved over long distances through the cytoplasm, and displayed in 64% of the cases an extremely bent shape, latter feature being the first time reported for Kip3 kinesins. Mitotic progression was delayed in the DkipB mutant and a higher number of cytoplasmic microtubules remained intact during mitosis. DkipB heterozygous strains showed an increased instability of diploid nuclei, which proved once more KipB involvement in mitosis, along with DkipB clear genetic interaction with a mutation in another mitotic kinesin in A. nidulans, bimC4. An N-terminal GFP-KipB construct localized to cytoplasmic microtubules in interphase cells and to spindle and astral microtubules during mitosis, in a discontinuous pattern. Speckles of GFP-KipB appeared to be aligned in the cell. Time-lapse video microscopy indicated that the spots were moving independently towards the microtubule plus ends. This advanced the hypothesis that KipB could display processivity and intrinsic motility along microtubules, or that other kinesins involved in organelle motility are able to target the KipB protein to the microtubule plus ends. In the case of C-terminally truncated GFP-KipB protein versions, a stronger GFP signal was obtained and colocalization with a-tubulin-GFP revealed that they uniformly stain cytoplasmic, mitotic and astral microtubules. This suggests that the C-terminus is important for the correct localization and the movement of KipB protein along microtubules

Towards plant species identification in complex samples: a bioinformatics pipeline for the identification of novel nuclear barcode candidates

Monitoring of the food chain to fight fraud and protect consumer health relies on the availability of methods to correctly identify the species present in samples, for which DNA barcoding is a promising candidate. The nuclear genome is a rich potential source of barcode targets, but has been relatively unexploited until now. Here, we show the development and use of a bioinformatics pipeline that processes available genome sequences to automatically screen large numbers of input candidates, identifies novel nuclear barcode targets and designs associated primer pairs, according to a specific set of requirements. We applied this pipeline to identify novel barcodes for plant species, a kingdom for which the currently available solutions are known to be insufficient. We tested one of the identified primer pairs and show its capability to correctly identify the plant species in simple and complex samples, validating the output of our approach.JRC.I.3-Molecular Biology and Genomic

Structural analysis reveals features of the spindle checkpoint kinase Bub1–kinetochore subunit Knl1 interaction

The function of the essential checkpoint kinases Bub1 and BubR1 requires their recruitment to mitotic kinetochores. Kinetochore recruitment of Bub1 and BubR1 is proposed to rely on the interaction of the tetratricopeptide repeats (TPRs) of Bub1 and BubR1 with two KI motifs in the outer kinetochore protein Knl1. We determined the crystal structure of the Bub1 TPRs in complex with the cognate Knl1 KI motif and compared it with the structure of the equivalent BubR1TPR–KI motif complex. The interaction developed along the convex surface of the TPR assembly. Point mutations on this surface impaired the interaction of Bub1 and BubR1 with Knl1 in vitro and in vivo but did not cause significant displacement of Bub1 and BubR1 from kinetochores. Conversely, a 62-residue segment of Bub1 that includes a binding domain for the checkpoint protein Bub3 and is C terminal to the TPRs was necessary and largely sufficient for kinetochore recruitment of Bub1. These results shed light on the determinants of kinetochore recruitment of Bub1

Bub1 regulates chromosome segregation in a kinetochore-independent manner

The kinetochore-bound protein kinase Bub1 performs two crucial functions during mitosis: it is essential for spindle checkpoint signaling and for correct chromosome alignment. Interestingly, Bub1 mutations are found in cancer tissues and cancer cell lines. Using an isogenic RNA interference complementation system in transformed HeLa cells and untransformed RPE1 cells, we investigate the effect of structural Bub1 mutants on chromosome segregation. We demonstrate that Bub1 regulates mitosis through the same mechanisms in both cell lines, suggesting a common regulatory network. Surprisingly, Bub1 can regulate chromosome segregation in a kinetochore-independent manner, albeit at lower efficiency. Its kinase activity is crucial for chromosome alignment but plays only a minor role in spindle checkpoint signaling. We also identify a novel conserved motif within Bub1 (amino acids 458–476) that is essential for spindle checkpoint signaling but does not regulate chromosome alignment, and we show that several cancer-related Bub1 mutants impair chromosome segregation, suggesting a possible link to tumorigenesis