NETWORKED2‐Subfamily Proteins Regulate the Cortical Actin Cytoskeleton of Growing Pollen Tubes and Polarised Pollen Tube Growth

We have recently characterised NET2A as a pollen‐specific actin‐binding protein which binds F‐actin at the plasma membrane of growing pollen tubes. However, the role of NET2 proteins in pollen development and fertilisation have yet to be elucidated. To further characterise the role of Arabidopsis NET2 proteins in pollen development and fertilisation, we analysed the subcellular localisation of NET2A over the course of pollen grain development, and investigated the role of the NET2 family using net2 loss‐of‐function mutants. We observed NET2A to localise to the F‐actin cytoskeleton in developing pollen grains as it underwent striking structural reorganisations at specific stages of development and during germination, and pollen tube growth. Furthermore, net2 loss‐of‐function mutants exhibited striking morphological defects in the early stages of pollen tube growth, arising from frequent alterations to pollen tube growth trajectory. We observed defects in the cortical actin cytoskeleton and actin‐driven subcellular processes in net2 mutant pollen tubes. We demonstrate that NET2 proteins are essential for normal actin‐driven pollen development highlighting an important role for the NET2 family members in regulating pollen tube growth during fertilisation

Inhibition of PFKFB3 Hampers the Progression of Atherosclerosis and Promotes Plaque Stability

Aims: 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB)3-mediated glycolysis is pivotal in driving macrophage- and endothelial cell activation and thereby inflammation. Once activated, these cells play a crucial role in the progression of atherosclerosis. Here, we analyzed the expression of PFKFB3 in human atherosclerotic lesions and investigated the therapeutic potential of pharmacological inhibition of PFKFB3 in experimental atherosclerosis by using the glycolytic inhibitor PFK158. Methods and Results: PFKFB3 expression was higher in vulnerable human atheromatous carotid plaques when compared to stable fibrous plaques and predominantly expressed in plaque macrophages and endothelial cells. Analysis of advanced plaques of human coronary arteries revealed a positive correlation of PFKFB3 expression with necrotic core area. To further investigate the role of PFKFB3 in atherosclerotic disease progression, we treated 6–8 weeks old male Ldlr–/– mice. These mice were fed a high cholesterol diet for 13 weeks, of which they were treated for 5 weeks with the glycolytic inhibitor PFK158 to block PFKFB3 activity. The incidence of fibrous cap atheroma (advanced plaques) was reduced in PFK158-treated mice. Plaque phenotype altered markedly as both necrotic core area and intraplaque apoptosis decreased. This coincided with thickening of the fibrous cap and increased plaque stability after PFK158 treatment. Concomitantly, we observed a decrease in glycolysis in peripheral blood mononuclear cells compared to the untreated group, which alludes that changes in the intracellular metabolism of monocyte and macrophages is advantageous for plaque stabilization. Conclusion: High PFKFB3 expression is associated with vulnerable atheromatous human carotid and coronary plaques. In mice, high PFKFB3 expression is also associated with a vulnerable plaque phenotype, whereas inhibition of PFKFB3 activity leads to plaque stabilization. This data implies that inhibition of inducible glycolysis may reduce inflammation, which has the ability to subsequently attenuate atherogenesis

Exo84c interacts with VAP27 to regulate exocytotic compartment degradation and stigma senescence

In plants, exocyst subunit isoforms exhibit significant functional diversity in that they are involved in either protein secretion or autophagy, both of which are essential for plant development and survival. Although the molecular basis of autophagy is widely reported, its contribution to plant reproduction is not very clear. Here, we have identified Exo84c, a higher plant-specific Exo84 isoform, as having a unique function in modulating exocytotic compartment degradation during stigmatic tissue senescence. This process is achieved through its interaction with the ER localised VAP27 proteins, which regulate the turnover of Exo84c through the autophagy pathway. VAP27 recruits Exo84c onto the ER membrane as well as numerous ER-derived autophagosomes that are labelled with ATG8. These Exo84c/exocyst and VAP27 positive structures are accumulated in the vacuole for degradation, and this process is partially perturbed in the exo84c knock-out mutants. Interestingly, the exo84c mutant showed a prolonged effective pollination period with higher seed sets, possibly because of the delayed stigmatic senescence when Exo84c regulated autophagy is blocked. In conclusion, our studies reveal a link between the exocyst complex and the ER network in regulating the degradation of exocytosis vesicles, a process that is essential for normal papilla cell senescence and flower receptivity

A phosphatidate phosphatase double mutant provides a new insight into plant membrane lipid homeostasis

Phospholipids make up the bulk of most eukaryotic cell membranes, but how their synthesis is regulated remains relatively poorly understood in plants. In our article1 we provide evidence that two Mg ( 2+) -dependent phosphatidic acid phosphatase enzymes, called PAH1 and PAH2, are capable of repressing phospholipid biosynthesis at the endoplasmic reticulum in Arabidopsis thaliana. The precise mechanism of repression remains unclear and it does appear to vary in several respects from that already described in Saccharomyces cerevisiae. ( 2,3)

Short-term regulation of hematopoiesis by lipoprotein(a) results in the production of pro-inflammatory monocytes

Background: Lipoproteins are important regulators of hematopoietic stem and progenitor cell (HSPC) biology, predominantly affecting myelopoiesis. Since myeloid cells, including monocytes and macrophages, promote the inflammatory response that propagates atherosclerosis, it is of interest whether the atherogenic low-density lipoprotein (LDL)-like particle lipoprotein(a) [Lp(a)] contributes to atherogenesis via stimulating myelopoiesis. Methods & results: To assess the effects of Lp(a)-priming on long-term HSPC behavior we transplanted BM of Lp(a) transgenic mice, that had been exposed to elevated levels of Lp(a), into lethally-irradiated C57Bl6 mice and hematopoietic reconstitution was analyzed. No differences in HSPC populations or circulating myeloid cells were detected ten weeks after transplantation. Likewise, in vitro stimulation of C57Bl6 BM cells for 24 h with Lp(a) did not affect colony formation, total cell numbers or myeloid populations 7 days later. To assess the effects of elevated levels of Lp(a) on myelopoiesis, C57Bl6 bone marrow (BM) cells were stimulated with lp(a) for 24 h, and a marked increase in granulocyte-monocyte progenitors, pro-inflammatory Ly6high monocytes and macrophages was observed. Seven days of continuous exposure to Lp(a) increased colony formation and enhanced the formation of pro-inflammatory monocytes and macrophages. Antibody-mediated neutralization of oxidized phospholipids abolished the Lp(a)-induced effects on myelopoiesis. Conclusion: Lp(a) enhances the production of inflammatory monocytes at the bone marrow level but does not induce cell-intrinsic long-term priming of HSPCs. Given the short-term and direct nature of this effect, we postulate that Lp(a)-lowering treatment has the capacity to rapidly revert this multi-level inflammatory response

TLC analysis of neutral lipids from castor developing male flowers and pollen.

<p>Lipid extracts were applied to a silica TLC plate which was developed with hexane/diethyl ether/acetic acid (70∶30∶1) before iodine-staining. The position of lipid standard components (lane N) and proposed nature of resolved sample lipids are shown. Lipid X and proposed sterol esters were purified for further analysis.</p

Candidate genes that may be important in tri-ricinolein synthesis based on RNA-Seq data.

<p>Candidate genes that may be important in tri-ricinolein synthesis based on RNA-Seq data.</p

Acyl-CoA levels in developing castor endosperm.

<p>The percentage of the total acyl-CoA peak area represented by specified acyl-CoAs are shown for castor endosperm stages during seed development. Analysis of fluorescent acyl-CoA derivatives was as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030100#pone.0030100-Larson1" target="_blank">[24]</a> and average values from three extractions and analyses for each stage are shown, together with the standard error in brackets (<i>n</i> = 3).</p>a<p>Endosperm samples were staged according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030100#pone.0030100-Greenwood1" target="_blank">[25]</a>.</p

Fatty acid analysis of castor tissues and purified lipid X.

<p>Average peak-area percentages of fatty acid methyl ester derivatives are listed. The number of separate sample extractions and analyses are listed in brackets, except for lipid X, where the result from two GC injections of the purified fraction is shown.</p>a<p>Italicised entries are probable fatty acid designations, although the species did not exactly co-chromatograph with standards.</p>b<p>Indicates the identity of the molecular species is not known but likely wax or hydroxylated fatty acid derivatives from pollen protective layers.</p