The endothelial cell spheroid assay provides a suitable in vitro model to study (lymph) angiogenesis and test pro- and anti-(lymph) angiogenic factors or drugs. Usually, the extent of cell invasion, observed through optical microscopy, is measured. The present study proposes the spatial distribution of migrated cells as a new descriptor of the (lymph) angiogenic response. The utility of this novel method rests with its capacity to locally characterise spheroid structure, allowing not only the investigation of single and collective cell invasion but also the evolution of the spheroid core itself. Moreover, the proposed method can be applied to 2D-projected spheroid images obtained by optical microscopy, as well as to 3D images acquired by confocal microscopy. To validate the proposed methodology, endothelial cell invasion was evaluated under different experimental conditions. The results were compared with widely used global parameters. The comparison shows that our method prevents local spheroid modifications from being overlooked and leading to the possible misinterpretation of results.

Kinesin motor proteins exert essential cellular functions in all eukaryotes. They control mitosis, migration and intracellular transport through interaction with microtubules. Small molecule inhibitors of the mitotic kinesin KiF11/Eg5 are a promising new class of anti-neoplastic agents currently evaluated in clinical cancer trials for solid tumors and hematological malignancies. Here we report induction of Eg5 and four other mitotic kinesins including KIF20A/Mklp2 upon stimulation of in vivo angiogenesis with vascular endothelial growth factor-A (VEGF-A). Expression analyses indicate up-regulation of several kinesin-encoding genes predominantly in lymphoblasts and endothelial cells. Chemical blockade of Eg5 inhibits endothelial cell proliferation and migration in vitro. Mitosis-independent vascular outgrowth in aortic ring cultures is strongly impaired after Eg5 or Mklp2 protein inhibition. In vivo, interfering with KIF11/Eg5 function causes developmental and vascular defects in zebrafish and chick embryos and potent inhibition of tumor angiogenesis in experimental tumor models. Besides blocking tumor cell proliferation, impairing endothelial function is a novel mechanism of action of kinesin inhibitors.

Lymphatic dysfunctions are associated with several human diseases, including lymphedema and metastatic spread of cancer. Although it is well recognized that lymphatic capillaries attach directly to interstitial matrix mainly composed of fibrillar type I collagen, the interactions occurring between lymphatics and their surrounding matrix have been overlooked. In this study, we demonstrate how matrix metalloproteinase (MMP)-2 drives lymphatic morphogenesis through Mmp2-gene ablation in mice, mmp2 knockdown in zebrafish and in 3D-culture systems, and through MMP2 inhibition. In all models used in vivo (3 murine models and thoracic duct development in zebrafish) and in vitro (lymphatic ring and spheroid assays), MMP2 blockage or down-regulation leads to reduced lymphangiogenesis or altered vessel branching. Our data show that lymphatic endothelial cell (LEC) migration through collagen fibers is affected by physical matrix constraints (matrix composition, density, and cross-linking). Transmission electron microscopy and confocal reflection microscopy using DQ-collagen highlight the contribution of MMP2 to mesenchymal-like migration of LECs associated with collagen fiber remodeling. Our findings provide new mechanistic insight into how LECs negotiate an interstitial type I collagen barrier and reveal an unexpected MMP2-driven collagenolytic pathway for lymphatic vessel formation and morphogenesis.

patients with ≥VGPR estimated to be 75. 8%. There were no significant differences between patients with a pre-transplant response ≥VGPR and partial response (PR) in terms of PFS (median 33 vs 24 months, p= 0. 3), TTP (not reached vs 25 months, p= 0. 2), TNT (not reached vs 33 months, p= 0. 09), and OS (not reached vs 71 months, p= 0. 8) (Figure 1). Conclusions. Patients with multiple myeloma who achieve PR or ≥VGPR before transplantation have similar disease outcomes. Since only 30% of patients will have a ≥VGPR with novel agents, at least PR is a reasonable pre-transplant treatment goal.

Cytokinins are involved in many aspects of plant growth and development, and physiological evidence also indicates that they have a role in floral transition. In order to integrate these phytohormones into the current knowledge of genetically defined molecular pathways to flowering, we performed exogenous treatments of adult wild type and mutant Arabidopsis plants, and analysed the expression of candidate genes. We used a hydroponic system that enables synchronous growth and flowering of Arabidopsis, and allows the precise application of chemicals to the roots for defined periods of time. We show that the application of N⁶-benzylaminopurine (BAP) promotes flowering of plants grown in non-inductive short days. The response to cytokinin treatment does not require FLOWERING LOCUS T (FT), but activates its paralogue TWIN SISTER OF FT (TSF), as well as FD, which encodes a partner protein of TSF, and the downstream gene SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1). Treatment of selected mutants confirmed that TSF and SOC1 are necessary for the flowering response to BAP, whereas the activation cascade might partially act independently of FD. These experiments provide a mechanistic basis for the role of cytokinins in flowering, and demonstrate that the redundant genes FT and TSF are differently regulated by distinct floral-inducing signals.

Dentin matrix protein 1 (DMP1) is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family, a group of proteins initially described as mineralized extracellular matrices components. More recently, SIBLINGs have been implicated in several key steps of cancer progression, including angiogenesis. Although proangiogenic activities have been demonstrated for 2 SIBLINGs, the role of DMP1 in angiogenesis has not yet been addressed. We demonstrate that this extracellular matrix protein induced the expression of vascular endothelial cadherin (VE-cadherin), a key regulator of intercellular junctions and contact inhibition of growth of endothelial cells that is also known to modulate vascular endothelial growth factor receptor 2 (VEGFR-2) activity, the major high-affinity receptor for VEGF. DMP1 induced VE-cadherin and p27(Kip1) expression followed by cell-cycle arrest in human umbilical vein endothelial cells (HUVECs) in a CD44-dependent manner. VEGF-induced proliferation, migration, and tubulogenesis responses were specifically blocked on DMP1 pretreatment of HUVECs. Indeed, after VE-cadherin induction, DMP1 inhibited VEGFR-2 phosphorylation and Src-mediated signaling. However, DMP1 did not interfere with basic fibroblast growth factor-induced angiogenesis. In vivo, DMP1 significantly reduced laser-induced choroidal neovascularization lesions and tumor-associated angiogenesis. These data enable us to put DMP1 on the angiogenic chessboard for the first time and to identify this protein as a new specific inhibitor of VEGF-induced angiogenesis.

Several tumor necrosis factor receptor (TNFR) family members activate both the classical and the alter- native NF- (cid:1) B pathways. However, how a single receptor engages these two distinct pathways is still poorly understood. Using lymphotoxin (cid:2) receptor (LT (cid:2) R) as a prototype, we showed that activation of the alternative, but not the classical, NF- (cid:1) B pathway relied on internalization of the receptor. Further molecular analyses revealed a specific cytosolic region of LT (cid:2) R essential for its internalization, TRAF3 recruitment, and p100 processing. Interestingly, we found that dynamin-dependent, internalization (cid:1) internalization mesenteric stromal of alternative NF- (cid:1) B on LT (cid:2) R cellular trafficking as a for specific biological functions of NF- (cid:1) immunoprecipitation experiment (1st IP) by incubation with either control beads or Flag-M2 beads. The immunoprecipitates were then used for detecting Flag-NIK and K48-linked polyubiquitinated NIK. Quantification of signals was per- formed with the Image J software. Oligomerization of LT (cid:2) R and GST pulldown. For studying the ability of LT (cid:3) R to form trimers, we performed transient transfections of HEK 293T cells with a combination of HA-, Flag-, and Myc-tagged LT (cid:3) R expression vectors. A first immunoprecipitation was performed using Flag-M2 agarose beads. The immunoprecipitated material was then selectively released overnight by compe- tition with an excess of 3 (cid:8) Flag peptide (Sigma). The supernatants were subjected to a second immunoprecipitation with either an anti-HA or an anti-Myc antibody for 2 h at 4°C. The immunoprecipitated materials were analyzed by immunoblotting with an anti-Myc or an anti-HA antibody, respectively. To an- alyze aggregation of ectopic LT (cid:3) R, HEK 293T cells were collected 40 h posttransfection, washed twice with phosphate-buffered saline (PBS), and then in- cubated for 30 min at room temperature in PBS containing 1 mM dithiobis[succinimidyl propionate] (DSP; Pierce/Thermo Fisher Scientific). After cross-linking, the cells were washed once in PBS and incubated for 15 min in 20 mM Tris-HCl (pH 7.4)–PBS to stop the reaction. The cells were scraped in SDS buffer, and protein extracts were analyzed by immunoblotting for LT (cid:3) R expression. pGEX-4T1-wt LT (cid:3) R or mutant expression vectors were transformed into Escherichia coli BL21. Bacterial cultures were grown to an A 600 of 0.6 and were induced with 0.5 mM isopropyl- (cid:3)

Several tumor necrosis factor receptor (TNFR) family members activate both the classical and the alternative NF-κB pathways. However, how a single receptor engages these two distinct pathways is still poorly understood. Using lymphotoxin β receptor (LTβR) as a prototype, we showed that activation of the alternative, but not the classical, NF-κB pathway relied on internalization of the receptor. Further molecular analyses revealed a specific cytosolic region of LTβR essential for its internalization, TRAF3 recruitment, and p100 processing. Interestingly, we found that dynamin-dependent, but clathrin-independent, internalization of LTβR appeared to be required for the activation of the alternative, but not the classical, NF-κB pathway. In vivo, ligand-induced internalization of LTβR in mesenteric lymph node stromal cells correlated with induction of alternative NF-κB target genes. Thus, our data shed light on LTβR cellular trafficking as a process required for specific biological functions of NF-κB.

Dentin matrix protein 1 (DMP1) is a member of the Small Integrin‐Binding LIgand N‐linked Glycoproteins (SIBLINGs) family, a group of proteins initially described as mineralized extracellular matrices components and more recently implicated in several key steps of cancer progression, including angiogenesis. Although pro‐angiogenic activities have been demonstrated for two SIBLINGs, the role of DMP1 in angiogenesis has not been addressed yet. We found that DMP1 inhibited the proliferation while it stimulated migration and tubulogenesis of human umbilical vein endothelial cells (HUVEC), thus promoting differentiation of endothelial cells. Further investigation of DMP1 mechanism of action evidenced a CD44‐dependent G1‐block and an increased p27Kip1 and VE‐cadherin expression in HUVEC.Our data identify DMP1 as a new regulator of angiogenesis that turns on the switch of the endothelial cell phenotype from proliferative to differentiated, through a CD44‐dependent differentiation program that links up with a VE‐cadherin pathway mediating contact inhibition of endothelial cell growth.

The generation of cortical projection neurons relies on the coordination of radial migration with branching. Here, we report that the multisubunit histone acetyltransferase Elongator complex, which contributes to transcript elongation, also regulates the maturation of projection neurons. Indeed, silencing of its scaffold (Elp1) or catalytic subunit (Elp3) cell-autonomously delays the migration and impairs the branching of projection neurons. Strikingly, neurons defective in Elongator show reduced levels of acetylated alpha-tubulin. Reduction of alpha-tubulin acetylation via expression of a nonacetylatable alpha-tubulin mutant leads to comparable defects in cortical neurons and suggests that alpha-tubulin is a target of Elp3. This is further supported by the demonstration that Elp3 promotes acetylation and counteracts HDAC6-mediated deacetylation of this substrate in vitro. Our results uncover alpha-tubulin as a target of the Elongator complex and suggest that a tight regulation of its acetylation underlies the maturation of cortical projection neurons.

Eukaryotic phosphomannomutases (PMMs) catalyze the interconversion of mannose 6-phosphate to mannose 1-phosphate and are essential to the biosynthesis of GDP-mannose. As such, plant PMMs are involved in ascorbic acid (AsA) biosynthesis and N-glycosylation. We report on the conditional phenotype of the temperature-sensitive Arabidopsis thaliana pmm- 12 mutant. Mutant seedlings were phenotypically similar to wild type seedlings when grown at 16 - 18 degrees C but died within several days after transfer to 28 degrees C. This phenotype was observed throughout both vegetative and reproductive development. Protein extracts derived from pmm-12 plants had lower PMM protein and enzyme activity levels. In vitro biochemical analysis of recombinant proteins showed that the mutant PMM protein was compromised in its catalytic efficiency (K-cat/K-m). Despite significantly decreased AsA levels in pmm-12 plants, AsA deficiency could not account for the observed phenotype. Since, at restrictive temperature, total glycoprotein patterns were altered and glycosylation of protein-disulfide isomerase was perturbed, we propose that a deficiency in protein glycosylation is responsible for the observed cell death phenotype.

During flowering, the CENTRORADIALIS ( CEN ) gene of Antirrhinum majus and its homolog TERMINAL FLOWER1 ( TFL1 ) in Arabidopsis ( Arabidopsis thaliana ) are required to maintain inflorescence identity of the shoot apical meristem (SAM) while flower meristems are produced on its flanks ([Bradley et

A single application of cytokinin benzyladenine causes a threefold increase in the frequency of plasmodesmata in the vegetative shoot apical meristem (SAM) of Sinapis alba plants. This increase is observed 20 h after application within all cell layers (L1, L2, L3) as well as at the interfaces between these layers. Evidence is presented indicating that cytokinin promotes mainly the formation of new secondary plasmodesmata. A similar increase in the frequency of secondary plasmodesmata was observed in the Sinapis SAM during the floral transition induced by a single long day, suggesting that this effect of the long day is mediated by cytokinin.

The long‐term response of Arabidopsis thaliana to increasing CO2 was evaluated in plants grown in 800 μl l−1 CO2 from sowing and maintained, in hydroponics, on three nitrogen supplies: “low,”“medium” and “high.” The global response to high CO2 and N‐supply was evaluated by measuring growth parameters in parallel with photosynthetic activity, leaf carbohydrates, ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) messenger RNA and protein, stomatal conductance (gs) and density. CO2 enrichment was found to stimulate biomass production, whatever the N‐supply. This stimulation was transient on low N‐supply and persisted throughout the whole vegetative growth only in high N‐supply. Acclimation on low N–high CO2 was not associated with carbohydrate accumulation or with a strong reduction in Rubisco amount or activity. At high N‐supply, growth stimulation by high CO2 was mainly because of the acceleration of leaf production and expansion while other parameters such as specific leaf area, root/shoot ratio and gs appeared to be correlated with total leaf area. Our results thus suggest that, in strictly controlled and stable growing conditions, acclimation of A. thaliana to long‐term CO2 enrichment is mostly controlled by growth rate adjustment.

BACKGROUND AND AIMS:Kip-related-proteins (KRPs), negative regulators of cell division, have recently been discovered in plants but their in planta function is as yet unclear. In this study the spatial expression of all seven KRP genes in shoot apices of Arabidopsis thaliana were compared.METHODS:In situ hybridization analyses were performed on longitudinal sections of shoot apices from 2-month-old Arabidopsis plants.KEY RESULTS:The study provides evidence for different expression pattern groups. KRP1 and KRP2 expression is restricted to the endoreduplicating tissues. In contrast, KRP4 and KRP5 expression is mainly restricted to mitotically dividing cells. KRP3, KRP6 and KRP7 can be found in both mitotically dividing and endoreduplicating cells.CONCLUSION:The results suggest differential roles for the distinct KRPs. KRP1 and KRP2 might specifically be involved in the establishment of polyploidy. In contrast, KRP4 and KRP5 might be involved in regulating the progression through the mitotic cell cycle. KRP3, KRP6 and KRP7 might have a function in both types of cell cycle.