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(A) cbk1-5E-E164S cbk1-aid cdc15-2 (YMF3905), (B) cbk1-5E-E251S cbk1-aid cdc15-2 (YMF3910), (C) cbk1-5E-E409S cbk1-aid cdc15-2 (YMF3995), (D) cbk1-5E-E615T cbk1-aid cdc15-2 (YMF3906), and (E) cbk1-5E-E711S cbk1-aid cdc15-2 (YMF3907) cells were grown in YPD and arrested in late anaphase by shifting the temperature to 37 °C before the addition of rapamycin to half of the culture for 20 min. To allow progression through the cell cycle, cells were released in the absence (i) or presence (ii) of rapamycin. Samples were taken at the indicated times to determine cell-cycle progression by flow cytometry. (F) 3D Cbk1 structure (PDB 4LQS [48]) in which different protein domains are highlighted. Residue T574 in the activation loop, together with residues D475 and S409 in the kinase domain are denoted. (G) cbk1-T574A cbk1-aid cdc15-2 cells (YMF4191) were grown as above. FACS graphs can be found in the supplementary FACS file (S1 File).

The indicated fungal species were compared by CLUSTAL O multiple sequence alignment. Kinase domain and activation loop are marked. Serines and threonines followed by proline in S. cerevisiae Cbk1 are indicated with an asterisk and the corresponding residue. Those residues were changed to glutamic acid to mimic phosphorylation (cbk1-6E) [42]. This mutant was used in this work.

(A) Schematic illustration of the “auxin inducible degron” system. (i) The Cbk1 auxin degron strain (cbk1-aid, where aid denotes the auxin inducible degron) contains a version of Cbk1 in which the auxin inducible degron was fused to the C-terminus of CBK1 in its own locus. Protein expression is under CBK1 promoter and Cbk1-aid is able to perform wt Cbk1’s functions. The fusion was carried out in a yeast strain in which the F-box protein Tir1 is expressed constitutively under the control of ADH1 promoter. Tir1 binds to SCF and forms the E3 ubiquitin ligase SCF-TIR1 that recruits the E2 ubiquitin conjugating enzyme [38]. (ii) Following the addition of auxins, the F-box Tir1 is able to specifically recognise the aid tag fused to Cbk1. Then, E2 ubiquitin conjugating enzyme polyubiquitylates aid, which rapidly promotes the degradation of Cbk1-aid by the proteasome (iii). This system allows to study the immediate biological consequences after Cbk1 depletion. (B) ADH-TIR1 (YJW15) and cbk1-aid ADH-TIR1 (YMF3657) cells were grown in YPD before the addition of NAA and IAA auxins. Samples were taken at the indicated times to determine cell-cycle progression by flow cytometry. (C) Schematic representation of experimental set-up in which cells were grown in YPD and arrested in late anaphase by shifting the temperature to 37 °C before the addition of NAA and IAA auxins for 50 min. Next, cells were incubated with DMSO (i) or rapamycin (ii) for 20 min while still arrested in anaphase. Cells were released in the absence (i) or presence (ii) of rapamycin, and with the NAA and IAA auxins present in medium throughout the rest of the experiment. (D) cbk1-aid cdc15-2 (YMF3866) cells were grown as represented in C. Samples were taken at shown times to determine cell-cycle progression by flow cytometry ((i) and (ii)) and cell morphology by light microscopy in the absence (iii) or in the presence (iv) of rapamycin at 120 min after the release from late anaphase arrest. Scale bars indicate 5

Tissue factor pathway inhibitor-alpha (TFPI-alpha) is a Kunitz-type serine protease inhibitor, which suppresses coagulation by inhibiting the tissue factor (TF)/factor VIIa complex as well as factor Xa. In static plasma-phospholipid systems, TFPI- thus suppresses both factor Xa and thrombin generation. In this article, we used a microfluidics approach to investigate how TFPI- regulates fibrin clot formation in platelet thrombi at low wall shear rate. We therefore hypothesized that the anticoagulant effect of TFPI-alpha in plasma is a function of the local procoagulant strengthdefined as the magnitude of thrombin generation under flow, due to local activities of TF/factor VIIa and factor Xa. To test this hypothesis, we modulated local coagulation by microspot coating of flow channels with 0 to 100 pM TF/collagen, or by using blood from patients with haemophilia A or B. For blood or plasma from healthy subjects, blocking of TFPI- enhanced fibrin formation, extending from a platelet thrombus, under flow only at <2 pM coated TF. This enhancement was paralleled by an increased thrombin generation. For mouse plasma, genetic deficiency in TFPI enhanced fibrin formation under flow also at 100 pM TF microspots. On the other hand, using blood from haemophilia A or B patients, TFPI-alpha antagonism markedly enhanced fibrin formation at microspots with up to 100 pM coated TF. We conclude that, under flow, TFPI-alpha is capable to antagonize fibrin formation in a manner dependent on and restricted by local TF/factor VIIa and factor Xa activities.

Tissue factor pathway inhibitor-alpha (TFPI-alpha) is a Kunitz-type serine protease inhibitor, which suppresses coagulation by inhibiting the tissue factor (TF)/factor VIIa complex as well as factor Xa. In static plasma-phospholipid systems, TFPI- thus suppresses both factor Xa and thrombin generation. In this article, we used a microfluidics approach to investigate how TFPI- regulates fibrin clot formation in platelet thrombi at low wall shear rate. We therefore hypothesized that the anticoagulant effect of TFPI-alpha in plasma is a function of the local procoagulant strengthdefined as the magnitude of thrombin generation under flow, due to local activities of TF/factor VIIa and factor Xa. To test this hypothesis, we modulated local coagulation by microspot coating of flow channels with 0 to 100 pM TF/collagen, or by using blood from patients with haemophilia A or B. For blood or plasma from healthy subjects, blocking of TFPI- enhanced fibrin formation, extending from a platelet thrombus, under flow only at <2 pM coated TF. This enhancement was paralleled by an increased thrombin generation. For mouse plasma, genetic deficiency in TFPI enhanced fibrin formation under flow also at 100 pM TF microspots. On the other hand, using blood from haemophilia A or B patients, TFPI-alpha antagonism markedly enhanced fibrin formation at microspots with up to 100 pM coated TF. We conclude that, under flow, TFPI-alpha is capable to antagonize fibrin formation in a manner dependent on and restricted by local TF/factor VIIa and factor Xa activities.

Ammonium nutrition often represents an important growth-limiting stress in plants. Some of the symptoms that plants present under ammonium nutrition have been associated with pH deregulation, in fact external medium pH control is known to improve plants ammonium tolerance. However, the way plant cell metabolism adjusts to these changes is not completely understood. Thus, in this work we focused on how Arabidopsis thaliana shoot and root respond to different nutritional regimes by varying the nitrogen source (NO3 and NH4), concentration (2 and 10 mM) and pH of the external medium (5.7 and 6.7) to gain a deeper understanding of cell metabolic adaptation upon altering these environmental factors. The results obtained evidence changes in the response of ammonium assimilation machinery and of the anaplerotic enzymes associated to Tricarboxylic Acids (TCA) cycle in function of the plant organ, the nitrogen source and the degree of ammonium stress. A greater stress severity at pH 5.7 was related to NH4 accumulation; this could not be circumvented in spite of the stimulation of glutamine synthetase, glutamate dehydrogenase, and TCA cycle anaplerotic enzymes. Moreover, this study suggests specific functions for different gin and gdh isoforms based on the nutritional regime. Overall, NH4 accumulation triggering ammonium stress appears to bear no relation to nitrogen assimilation impairment.

Ammonium nutrition often represents an important growth-limiting stress in plants. Some of the symptoms that plants present under ammonium nutrition have been associated with pH deregulation, in fact external medium pH control is known to improve plants ammonium tolerance. However, the way plant cell metabolism adjusts to these changes is not completely understood. Thus, in this work we focused on how Arabidopsis thaliana shoot and root respond to different nutritional regimes by varying the nitrogen source (NO3 and NH4), concentration (2 and 10 mM) and pH of the external medium (5.7 and 6.7) to gain a deeper understanding of cell metabolic adaptation upon altering these environmental factors. The results obtained evidence changes in the response of ammonium assimilation machinery and of the anaplerotic enzymes associated to Tricarboxylic Acids (TCA) cycle in function of the plant organ, the nitrogen source and the degree of ammonium stress. A greater stress severity at pH 5.7 was related to NH4 accumulation; this could not be circumvented in spite of the stimulation of glutamine synthetase, glutamate dehydrogenase, and TCA cycle anaplerotic enzymes. Moreover, this study suggests specific functions for different gin and gdh isoforms based on the nutritional regime. Overall, NH4 accumulation triggering ammonium stress appears to bear no relation to nitrogen assimilation impairment.

Ammonium nutrition often represents an important growth-limiting stress in plants. Some of the symptoms that plants present under ammonium nutrition have been associated with pH deregulation, in fact external medium pH control is known to improve plants ammonium tolerance. However, the way plant cell metabolism adjusts to these changes is not completely understood. Thus, in this work we focused on how Arabidopsis thaliana shoot and root respond to different nutritional regimes by varying the nitrogen source (NO3 and NH4), concentration (2 and 10 mM) and pH of the external medium (5.7 and 6.7) to gain a deeper understanding of cell metabolic adaptation upon altering these environmental factors. The results obtained evidence changes in the response of ammonium assimilation machinery and of the anaplerotic enzymes associated to Tricarboxylic Acids (TCA) cycle in function of the plant organ, the nitrogen source and the degree of ammonium stress. A greater stress severity at pH 5.7 was related to NH4 accumulation; this could not be circumvented in spite of the stimulation of glutamine synthetase, glutamate dehydrogenase, and TCA cycle anaplerotic enzymes. Moreover, this study suggests specific functions for different gin and gdh isoforms based on the nutritional regime. Overall, NH4 accumulation triggering ammonium stress appears to bear no relation to nitrogen assimilation impairment.

Noccaea caerulescens (Nc) exhibits a very high constitutive expression of the heavy metal transporting ATPase, HMA4, as compared to the non-hyperaccumulator Arabidopsis thaliana (At), due to copy number expansion and altered cis-regulation. We screened a BAC library for HMA4 and found that HMA4 is triplicated in the genome of a N. caerulescens accession from a former Zn mine near La Calamine (LC), Belgium. We amplified multiple HMA4 promoter sequences from three calamine N. caerulescens accessions, and expressed AtHMA4 and different NcHMA4 cDNAs under At and Nc HMA4 promoters in the A. thaliana (Col) hma2hma4 double mutant. Transgenic lines expressing HMA4 under the At promoter were always fully complemented for root-toshoot Zn translocation and developed normally at a 2-mu M Zn supply, whereas the lines expressing HMA4 under Nc promoters usually showed only slightly enhanced root to shoot Zn translocation rates in comparison with the double mutant, probably owing to ectopic expression in the roots, respectively. When expression of the Zn deficiency responsive marker gene ZIP4 was tested, the transgenic lines expressing AtHMA4 under an NcHMA4-1-LC promoter showed on average a 7-fold higher expression in the leaves, in comparison with the double hma2hma4 mutant, showing that this construct aggravated, rather than alleviated the severity of foliar Zn deficiency in the mutant, possible owing to expression in the leaf mesophyll.