Apical myosin XI anticipates F‐actin during polarized growth of Physcomitrella patens cells

Tip growth is essential for land colonization by bryophytes, plant sexual reproduction and water and nutrient uptake. Because this specialized form of polarized cell growth requires both a dynamic actin cytoskeleton and active secretion, it has been proposed that the F‐actin‐associated motor myosin XI is essential for this process. Nevertheless, a spatial and temporal relationship between myosin XI and F‐actin during tip growth is not known in any plant cell. Here, we use the highly polarized cells of the moss Physcomitrella patens to show that myosin XI and F‐actin localize, in vivo, at the same apical domain and that both signals fluctuate. Surprisingly, phase analysis shows that increase in myosin XI anticipates that of F‐actin; in contrast, myosin XI levels at the tip fluctuate in identical phase with a vesicle marker. Pharmacological analysis using a low concentration of the actin polymerization inhibitor latrunculin B showed that the F‐actin at the tip can be significantly diminished while myosin XI remains elevated in this region, suggesting that a mechanism exists to cluster myosin XI‐associated structures at the cell's apex. In addition, this approach uncovered a mechanism for actin polymerization‐dependent motility in the moss cytoplasm, where myosin XI‐associated structures seem to anticipate and organize the actin polymerization machinery. From our results, we inferred a model where the interaction between myosin XI‐associated vesicular structures and F‐actin polymerization‐driven motility function at the cell's apex to maintain polarized cell growth. We hypothesize this is a general mechanism for the participation of myosin XI and F‐actin in tip growing cells.     

Oryza sativa Lysine‐Histidine‐type Transporter 1 functions in root uptake and root‐to‐shoot allocation of amino acids in rice

In agricultural soils, amino acids can represent vital nitrogen (N) sources for crop growth and yield. However, the molecular mechanisms underlying amino acid uptake and allocation are poorly understood in crop plants. This study shows that rice (Oryza sativa L.) roots can acquire aspartate at soil concentration, and that japonica subspecies take up this acidic amino acid 1.5‐fold more efficiently than indica subspecies. Genetic association analyses with 68 representative japonica or indica germplasms identified rice Lysine‐Histidine‐type Transporter 1 (OsLHT1) as a candidate gene associated with the aspartate uptake trait. When expressed in yeast, OsLHT1 supported cell growth on a broad spectrum of amino acids, and effectively transported aspartate, asparagine and glutamate. OsLHT1 is localized throughout the rice root, including root hairs, epidermis, cortex and stele, and to the leaf vasculature. Knockout of OsLHT1 in japonica resulted in reduced root uptake of amino acids. Furthermore, in ¹⁵N‐amino acid‐fed mutants versus wild‐type, a higher percentage of ¹⁵N remained in roots instead of being allocated to the shoot. ¹⁵N‐ammonium uptake and subsequently the delivery of root‐synthesized amino acids to Oslht1 shoots were also significantly decreased, which was accompanied by reduced shoot growth. These results together provide evidence that OsLHT1 functions in both root uptake and root to shoot allocation of a broad spectrum of amino acids in rice.     

Multi‐step down‐regulation of the secretory pathway in mitosis: A fresh perspective on protein trafficking

The secretory pathway delivers proteins synthesized at the rough endoplasmic reticulum (RER) to various subcellular locations via the Golgi apparatus. Currently, efforts are focused on understanding the molecular machineries driving individual processes at the RER and Golgi that package, modify and transport proteins. However, studies are routinely performed using non‐dividing cells. This obscures the critical issue of how the secretory pathway is affected by cell division. Indeed, several studies have indicated that protein trafficking is down‐regulated during mitosis. Moreover, the RER and Golgi apparatus exhibit gross reorganization in mitosis. Here I provide a relatively neglected perspective of how the mitotic cyclin‐dependent kinase (CDK1) could regulate various stages of the secretory pathway. I highlight several aspects of the mitotic control of protein trafficking that remain unresolved and suggest that further studies on how the mitotic CDK1 influences the secretory pathway are necessary to obtain a deeper understanding of protein transport.     

Effects of inositol 1,4,5-trisphosphate on calcium release from the endoplasmic reticulum and Golgi apparatus in mouse mammary epithelial cells: a comparison during pregnancy and lactation

It has been established that inositol 1,4,5-trisphosphate(IP3) is responsible for the mobilization of calcium(Ca2+) from intracellular locations in a wide variety of tissues, and that this response triggers the stimulation of several hormones and neurotransmitters. However, these phenomena have yet to be examined in the mammary epithelium. Ca2+ uptake from the medium into the endoplasmic reticulum(ER) and Golgi apparatus in vitro in both pregnant and lactating mouse mammary epithelial cells was studied and a strong Ca2+ release from these organelles into the medium with the use of IP3 was shown. The Ca2+ uptake and its release due to IP3 was also usually greater during pregnancy than lactation.     

The oxalyl‐CoA synthetase‐regulated oxalate and its distinct effects on resistance to bacterial blight and aluminium toxicity in rice

• Oxalic acid is widely distributed in biological systems and known to play functional roles in plants. The gene AAE3 was recently identified to encode an oxalyl‐CoA synthetase (OCS) in Arabidopsis that catalyses the conversion of oxalate and CoA into oxalyl‐CoA. It will be particularly important to characterise the homologous gene in rice since rice is not only a monocotyledonous model plant, but also a staple food crop.
• Various enzymatic and biological methods have been used to characterise the homologous gene.
• We first defined that AAE3 in the rice genome (OsAAE3) also encodes an OCS enzyme. Its K_m for oxalate is 1.73 ± 0.12 mm , and V_m is 6824.9 ± 410.29 U·min^?1·mg protein^?1. Chemical modification and site‐directed mutagenesis analyses identified thiols as the active site residues for rice OCS catalysis, suggesting that the enzyme might be regulated by redox state. Subcellular localisation assay showed that the enzyme is located in the cytosol and predominantly distributed in leaf epidermal cells. As expected, oxalate levels increased when OCS was suppressed in RNAi transgenic plants. More interestingly, OCS‐suppressed plants were more susceptible to bacterial blight but more resistant to Al toxicity.
• The results demonstrate that the OsAAE3‐encoded protein also acts as an OCS in rice, and may play different roles in coping with stresses. These molecular, enzymatic and functional data provide first‐hand information to further clarify the function and mechanism of OCS in rice plants.

     

Dwarf and deformed flower 1, encoding an F‐box protein,is critical for vegetative and floral development in rice (Oryza sativa L.)

Recent studies have shown that F‐box proteins constitute a large family in eukaryotes, and play pivotal roles in regulating various developmental processes in plants. However, their functions in monocots are still obscure. In this study, we characterized a recessive mutant dwarf and deformed flower 1‐1 (ddf1‐1) in Oryza sativa (rice). The mutant is abnormal in both vegetative and reproductive development, with significant size reduction in all organs except the spikelet. DDF1 controls organ size by regulating both cell division and cell expansion. In the ddf1‐1 spikelet, the specification of floral organs in whorls 2 and 3 is altered, with most lodicules and stamens being transformed into glume‐like organs and pistil‐like organs, respectively, but the specification of lemma/palea and pistil in whorls 1 and 4 is not affected. DDF1 encodes an F‐box protein anchored in the nucleolus, and is expressed in almost all vegetative and reproductive tissues. Consistent with the mutant floral phenotype, DDF1 positively regulates B‐class genes OsMADS4 and OsMADS16, and negatively regulates pistil specification gene DL. In addition, DDF1 also negatively regulates the Arabidopsis LFY ortholog APO2, implying a functional connection between DDF1 and APO2. Collectively, these results revealed that DDF1, as a newly identified F‐box gene, is a crucial genetic factor with pleiotropic functions for both vegetative growth and floral organ specification in rice. These findings provide additional insights into the molecular mechanism controlling monocot vegetative and reproductive development.     

2‐phenylethynesulphonamide (PFT‐μ) enhances the anticancer effect of the novel hsp90 inhibitor NVP‐AUY922 in melanoma,by reducing GSH levels

Heat shock proteins (HSPs), are molecular chaperones that assist the proper folding of nascent proteins. This study aims to evaluate the antitumour effects of the hsp90 inhibitor NVP‐AUY922 in melanoma, both in vitro and in vivo. Our results show that NVP‐AUY922 inhibits melanoma cell growth in vitro, with down regulation of multiple signalling pathways involved in melanoma progression such as NF‐?B and MAPK/ERK. However, NVP‐AUY922 was unable to limit tumour growth in vivo. Cotreatment of A375M xenografts with NVP‐AUY922 and PFT‐μ, a dual inhibitor of both hsp70 and autophagy, induced a synergistic increase of cell death in vitro, and delayed tumour formation in A375M xenografts. PFT‐μ depleted cells from the reduced form of glutathione (GSH) and increased oxidative stress. The oxidative stress induced by PFT‐μ further enhanced NVP‐AUY922‐induced cytotoxic effects. These data suggest a potential therapeutic role for NVP‐AUY922 used in combination with PFT‐μ, in melanoma.     

Calcium acetate‐induced reduction of cadmium accumulation in Oryza sativa: Expression of auto‐inhibited calcium‐ATPase and cadmium transporters

• Calcium (Ca) signalling has an essential role in regulating plant responses to various abiotic stresses.
• This study applied Ca in various forms (Ca acetate and CaCl₂) and concentrations to reduce cadmium (Cd) concentration in rice and propose a possible mechanism through which Ca acts to control the Cd concentration in rice.
• The results showed that supplementation of Cd‐contaminated soil with Ca acetate reduced the Cd concentration in rice after exposure for 7 days in both hydroponic and soil conditions. The possible involvement of the auto‐inhibited Ca²⁺‐ATPase gene (ACA) might act to control the primary signal of the Cd stress response. The messages from ACA3 and ACA13 tended to up‐regulate the low‐affinity cation transporter (OsLCT1) and down‐regulate Cd uptake and the Cd translocation transporter, including the genes, natural resistance‐associated macrophage protein 5 (Nramp5) and Zn/Cd‐transporting ATPase 2 (HMA2), which resulted in a reduction in the Cd concentration in rice. After cultivation for 120 days, the application of Ca acetate into Cd‐contaminated soil inhibited Cd uptake of rice.
• Increasing the Ca acetate concentration in the soil lowered the Cd concentration in rice shoots and grains. Moreover, Ca acetate maintained rice productivity and quality whereas both aspects decreased under Cd stress.

     

Chemical screening identifies ROCK as a target for recovering mitochondrial function in Hutchinson‐Gilford progeria syndrome

Hutchinson‐Gilford progeria syndrome (HGPS) constitutes a genetic disease wherein an aging phenotype manifests in childhood. Recent studies indicate that reactive oxygen species (ROS) play important roles in HGPS phenotype progression. Thus, pharmacological reduction in ROS levels has been proposed as a potentially effective treatment for patient with this disorder. In this study, we performed high‐throughput screening to find compounds that could reduce ROS levels in HGPS fibroblasts and identified rho‐associated protein kinase (ROCK) inhibitor (Y‐27632) as an effective agent. To elucidate the underlying mechanism of ROCK in regulating ROS levels, we performed a yeast two‐hybrid screen and discovered that ROCK1 interacts with Rac1b. ROCK activation phosphorylated Rac1b at Ser71 and increased ROS levels by facilitating the interaction between Rac1b and cytochrome c. Conversely, ROCK inactivation with Y‐27632 abolished their interaction, concomitant with ROS reduction. Additionally, ROCK activation resulted in mitochondrial dysfunction, whereas ROCK inactivation with Y‐27632 induced the recovery of mitochondrial function. Furthermore, a reduction in the frequency of abnormal nuclear morphology and DNA double‐strand breaks was observed along with decreased ROS levels. Thus, our study reveals a novel mechanism through which alleviation of the HGPS phenotype is mediated by the recovery of mitochondrial function upon ROCK inactivation.     

KITLG is a novel target of miR‐34c that is associated with the inhibition of growth and invasion in colorectal cancer cells

MiR‐34c is considered a potent tumour suppressor because of its negative regulation of multiple target mRNAs that are critically associated with tumorigenesis and metastasis. In the present study, we demonstrated a novel target of miR‐34c, KITLG, which has been implicated in colorectal cancer (CRC). First, we found a significant negative relationship between miR‐34c and KITLG mRNA expression levels in CRC cell lines, including HT‐29, HCT‐116, SW480 and SW620 CRC cell lines. In silico analysis predicted putative binding sites for miR‐34c in the 3′ untranslated region (3′UTR) of KITLG mRNA. A dual‐luciferase reporter assay further confirmed that KITLG is a direct target of miR‐34c. Then, the cell lines were infected with lentiviruses expressing miR‐34c or a miR‐34c specific inhibitor. Restoration of miR‐34c dramatically reduced the expression of KITLG mRNA and protein, while silencing of endogenous miR‐34c increased the expression of KITLG protein. The miR‐34c‐mediated down‐regulation of KITLG was associated with the suppression on proliferation, cellular transformation, migration and invasion of CRC cells, as well as the promotion on apoptosis. Knockdown of KITLG by its specific siRNA confirmed a critical role of KITLG down‐regulation for the tumour‐suppressive effects of miR‐34c in CRC cells. In conclusion, our results demonstrated that miR‐34c might interfere with KITLG‐related CRC and could be a novel molecular target for CRC patients.     

Identification and molecular mapping of indica high‐tillering dwarf mutant htd4, a mild phenotype allelic mutant of D14 in rice (Oryza sativa L.)

• Metabolism of strigolactones (SLs) can improve the efficiency of nutrient use by regulating the development of roots and shoots in crops, making them an important research focus for molecular breeding. However, as a very important plant hormone, the molecular mechanism of SL signal transduction still remains largely unknown.
• In this study, we isolated an indica high‐tillering dwarf mutant 4 (htd4), a spontaneous mutant of rice, from the restorer line Gui99.
• Mapping and sequencing analysis showed that htd4 was a novel allelic mutant of D14, in which a single base substitution forms a premature termination codon. Quantitative RT‐PCR analyses revealed that expression levels of the genes D10, D17, D27, D3 and D14 increased significantly, while expression of D53 decreased in htd4, compared with the wild type. A subcellular localisation assay showed that the mutant of D14 in htd4 did not disturb the normal localisation of D14 proteins. However, a BiFC assay suggested that the mutant‐type D14 could not interact with D3. Additionally, compared with other D14 allelic mutants, htd4 was the first mutant of D14 discovered in indica, and the differences in many yield traits such as plant height, seed‐setting rate and grain sizes between htd4 and the wild type were less than those between other D14 allelic mutants and the wild type.
• Therefore, htd4 is considered a mild phenotype allelic mutant of D14. We conclude that the absence of functional D14 caused the high‐tillering dwarf phenotype of htd4. Our results may provide vital information for research on D14 function and the application of htd4 in molecular breeding.

     

High levels of Mn2+ inhibit secretory pathway Ca2+/Mn2+‐ATPase (SPCA) activity and cause Golgi fragmentation in neurons and glia

Excess Mn²⁺ in humans causes a neurological disorder known as manganism, which shares symptoms with Parkinson's disease. However, the cellular mechanisms underlying Mn²⁺‐neurotoxicity and the involvement of Mn²⁺‐transporters in cellular homeostasis and repair are poorly understood and require further investigation. In this work, we have analyzed the effect of Mn²⁺ on neurons and glia from mice in primary cultures. Mn²⁺ overload compromised survival of both cell types, specifically affecting cellular integrity and Golgi organization, where the secretory pathway Ca²⁺/Mn²⁺‐ATPase is localized. This ATP‐driven Mn²⁺ transporter might take part in Mn²⁺ accumulation/detoxification at low loads of Mn²⁺, but its ATPase activity is inhibited at high concentration of Mn²⁺. Glial cells appear to be significantly more resistant to this toxicity than neurons and their presence in cocultures provided some protection to neurons against degeneration induced by Mn²⁺. Interestingly, the Mn²⁺ toxicity was partially reversed upon Mn²⁺ removal by wash out or by the addition of EDTA as a chelating agent, in particular in glial cells. These studies provide data on Mn²⁺ neurotoxicity and may contribute to explore new therapeutic approaches for reducing Mn²⁺ poisoning.     

Co‐recruitment analysis of the CBL and CBLB signalosomes in primary T cells identifies CD5 as a key regulator of TCR‐induced ubiquitylation

T‐cell receptor (TCR) signaling is essential for the function of T cells and negatively regulated by the E3 ubiquitin–protein ligases CBL and CBLB. Here, we combined mouse genetics and affinity purification coupled to quantitative mass spectrometry to monitor the dynamics of the CBL and CBLB signaling complexes that assemble in normal T cells over 600 seconds of TCR stimulation. We identify most previously known CBL and CBLB interacting partners, as well as a majority of proteins that have not yet been implicated in those signaling complexes. We exploit correlations in protein association with CBL and CBLB as a function of time of TCR stimulation for predicting the occurrence of direct physical association between them. By combining co‐recruitment analysis with biochemical analysis, we demonstrated that the CD5 transmembrane receptor constitutes a key scaffold for CBL‐ and CBLB‐mediated ubiquitylation following TCR engagement. Our results offer an integrated view of the CBL and CBLB signaling complexes induced by TCR stimulation and provide a molecular basis for their negative regulatory function in normal T cells.