Antigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show that Ki-67 controls heterochromatin organisation. Altering Ki-67 expression levels did not significantly affect cell proliferation in vivo. Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently. Conversely, upregulation of Ki-67 expression in differentiated tissues did not prevent cell cycle arrest. Ki-67 interactors included proteins involved in nucleolar processes and chromatin regulators. Ki-67 depletion disrupted nucleologenesis but did not inhibit pre-rRNA processing. In contrast, it altered gene expression. Ki-67 silencing also had wide-ranging effects on chromatin organisation, disrupting heterochromatin compaction and long-range genomic interactions. Trimethylation of histone H3K9 and H4K20 was relocalised within the nucleus. Finally, overexpression of human or Xenopus Ki-67 induced ectopic heterochromatin formation. Altogether, our results suggest that Ki-67 expression in proliferating cells spatially organises heterochromatin, thereby controlling gene expression.DOI: http://dx.doi.org/10.7554/eLife.13722.001

Aquaporins form a family of water and solute channel proteins and are present in most living organisms. In plants, aquaporins play an important role in the regulation of root water transport in response to abiotic stresses. In this work, we investigated the role of phosphorylation of plasma membrane intrinsic protein (PIP) aquaporins in the Arabidopsis thaliana root by a combination of quantitative mass spectrometry and cellular biology approaches. A novel phosphoproteomics procedure that involves plasma membrane purification, phosphopeptide enrichment with TiO 2 columns, and systematic mass spectrometry sequencing revealed multiple and adjacent phosphorylation sites in the C-terminal tail of several AtPIPs. Six of these sites had not been de- Aquaporins form a family of channel proteins that mediate the transport across membranes of water, small neutral solutes, and occasionally ions (1-3). Aquaporins are present in all living kingdoms and in plants. Aquaporins exhibit a characteristically high multiplicity of forms with for instance 35 members in Arabidopsis (4, 5). Based upon their amino acid sequence homology, plant aquaporins can be classified into four subfamilies (4 -6). One of these corresponds to the plasma membrane intrinsic proteins (PIPs).1 The PIPs with 13 members in Arabidopsis represent the most abundant aquaporins in the plasma membrane (PM) and can be further divided into two sequence homology groups (AtPIP1 and AtPIP2). Aquaporins are 25-35-kDa proteins that share a typical organization with six transmembrane ␣-helices interrupted by five connecting loops (loops A-E) (7,8). In PM aquaporins, the N and C termini as well as loops B and D are exposed in the cytosol, whereas loops A, C, and E face the cell wall.Plants need to continuously adjust their water status in response to changing environmental conditions, and aquaporins play an important role in these processes (3, 9, 10). In particular, physiological and genetics studies have provided compelling evidence for a role of aquaporins in the regulation, in response to abiotic stresses, of root water transport, i.e. root hydraulic conductivity (Lp r ) (10, 11). For instance, exposure of Arabidopsis plants to salt (100 mM NaCl) induced a rapid (half-time, 45 min) and significant decrease (Ϫ70%) in Lp r that was maintained for at least 24 h (12). Whereas the long term effect of this NaCl stress can be accounted for by an overall transcriptional downregulation of aquaporins, the molecular mechanisms involved in the early inhibition of Lp r by NaCl are not fully understood yet. These mechanisms involve a slight decrease in overall abundance of AtPIP1 proteins as soon as 30 min after exposure to NaCl and a trafficking of AtPIP1 and AtPIP2 isoforms between the PM and intracellular compartments that may contribute to reducing the abundance of AtPIPs at the PM and therefore the hydraulic conductivity of salt-stressed root cells (12). Chilling is another stress that leads to inhibition of Lp r , and a relationship From the ‡Biochimie et Physiologie Molé c...

Leishmania is exposed to a sudden increase in environmental temperature during the infectious cycle that triggers stage differentiation and adapts the parasite phenotype to intracellular survival in the mammalian host. The absence of classical promoter-dependent mechanisms of gene regulation and constitutive expression of most of the heat-shock proteins (HSPs) in these human pathogens raise important unresolved questions as to regulation of the heat-shock response and stage-specific functions of Leishmania HSPs. Here we used a gel-based quantitative approach to assess the Leishmania donovani phosphoproteome and revealed that 38% of the proteins showed significant stage-specific differences, with a strong focus of amastigote-specific phosphoproteins on chaperone function. We identified STI1/HOP-containing chaperone complexes that interact with ribosomal client proteins in an amastigote-specific manner. Genetic analysis of STI1/HOP phosphorylation sites in conditional sti1

Nutritional symbiotic interactions require the housing of large numbers of microbial symbionts, which produce essential compounds for the growth of the host. In the legume-rhizobium nitrogen-fixing symbiosis, thousands of rhizobium microsymbionts, called bacteroids, are confined intracellularly within highly specialized symbiotic host cells. In Inverted Repeat-Lacking Clade (IRLC) legumes such as Medicago spp., the bacteroids are kept under control by an arsenal of nodulespecific cysteine-rich (NCR) peptides, which induce the bacteria in an irreversible, strongly elongated, and polyploid state. Here, we show that in Aeschynomene spp. legumes belonging to the more ancient Dalbergioid lineage, bacteroids are elongated or spherical depending on the Aeschynomene spp. and that these bacteroids are terminally differentiated and polyploid, similar to bacteroids in IRLC legumes. Transcriptome, in situ hybridization, and proteome analyses demonstrated that the symbiotic cells in the Aeschynomene spp. nodules produce a large diversity of NCR-like peptides, which are transported to the bacteroids. Blocking NCR transport by RNA interference-mediated inactivation of the secretory pathway inhibits bacteroid differentiation. Together, our results support the view that bacteroid differentiation in the Dalbergioid clade, which likely evolved independently from the bacteroid differentiation in the IRLC clade, is based on very similar mechanisms used by IRLC legumes.

The water status of plant leaves depends on the efficiency of the water supply, from the vasculature to inner tissues. This process is under hormonal and environmental regulation and involves aquaporin water channels. In Arabidopsis thaliana, the rosette hydraulic conductivity (K ros ) is higher in darkness than it is during the day. Knockout plants showed that three plasma membrane intrinsic proteins (PIPs) sharing expression in veins (PIP1;2, PIP2;1, and PIP2;6) contribute to rosette water transport, and PIP2;1 can fully account for K ros responsiveness to darkness. Directed expression of PIP2;1 in veins of a pip2;1 mutant was sufficient to restore K ros . In addition, a positive correlation, in both wild-type and PIP2;1-overexpressing plants, was found between K ros and the osmotic water permeability of protoplasts from the veins but not from the mesophyll. Thus, living cells in veins form a major hydraulic resistance in leaves. Quantitative proteomic analyses showed that light-dependent regulation of K ros is linked to diphosphorylation of PIP2;1 at Ser-280 and Ser-283. Expression in pip2;1 of phosphomimetic and phosphorylation-deficient forms of PIP2;1 demonstrated that phosphorylation at these two sites is necessary for K ros enhancement under darkness. These findings establish how regulation of a single aquaporin isoform in leaf veins critically determines leaf hydraulics.

No abstract

Accurate chromosome segregation relies upon a mitotic checkpoint that monitors kinetochore attachment toward opposite spindle poles before enabling chromosome disjunction [1]. The MPS1/TTK protein kinase is a core component of the mitotic checkpoint that lies upstream of MAD2 and BubR1 both at the kinetochore and in the cytoplasm [2, 3]. To gain insight into the mechanisms underlying the regulation of MPS1 kinase, we undertook the identification of Xenopus MPS1 phosphorylation sites by mass spectrometry. We mapped several phosphorylation sites onto MPS1 and we show that phosphorylation of S283 in the noncatalytic region of MPS1 is required for full kinase activity. This phosphorylation potentiates MPS1 catalytic efficiency without impairing its affinity for the substrates. By using Xenopus egg extracts depleted of endogenous MPS1 and reconstituted with single point mutants, we show that phosphorylation of S283 is essential to activate the mitotic checkpoint. This phosphorylation does not regulate the localization of MPS1 to the kinetochore but is required for the recruitment of MAD1/MAD2, demonstrating its role at the kinetochore. Constitutive phosphorylation of S283 lowers the number of kinetochores required to hold the checkpoint, which suggests that CDK-dependent phosphorylation of MPS1 is essential to sustain the mitotic checkpoint when few kinetochores remain unattached.

Iron (Fe) is a major micronutrient and is required for plant growth and development. Nongrass species have evolved a reduction-based strategy to solubilize and take up Fe. The secretion of Fe-mobilizing coumarins (e.g. fraxetin, esculetin and sideretin) by plant roots plays an important role in this process. Although the biochemical mechanisms leading to their biosynthesis have been well described, very little is known about their cellular and subcellular localization or their mobility within plant tissues. Spectral imaging was used to monitor, in Arabidopsis thaliana, the in planta localization of Fe-mobilizing coumarins and scopolin. Molecular, genetic and biochemical approaches were also used to investigate the dynamics of coumarin accumulation in roots. These approaches showed that root hairs play a major role in scopoletin secretion, whereas fraxetin and esculetin secretion occurs through all epidermis cells. The findings of this study also showed that the transport of coumarins from the cortex to the rhizosphere relies on the PDR9 transporter under Fe-deficient conditions. Additional experiments support the idea that coumarins move throughout the plant body via the xylem sap and that several plant species can take up coumarins present in the surrounding media. Altogether, the data presented here demonstrate that coumarin storage and accumulation in roots is a highly complex and dynamic process.