NPC1-mTORC1 Signaling Couples Cholesterol Sensing to Organelle Homeostasis and Is a Targetable Pathway in Niemann-Pick Type C

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The R-Ras/RIN2/Rab5 complex controls endothelial cell adhesion and morphogenesis via active integrin endocytosis and Rac signaling

During developmental and tumor angiogenesis, semaphorins regulate blood vessel navigation by signaling through plexin receptors that inhibit the R-Ras subfamily of small GTPases. R-Ras is mainly expressed in vascular cells, where it induces adhesion to the extracellular matrix (ECM) through unknown mechanisms. We identify the Ras and Rab5 interacting protein RIN2 as a key effector that in endothelial cells interacts with and mediates the pro-adhesive and -angiogenic activity of R-Ras. Both R-Ras-GTP and RIN2 localize at nascent ECM adhesion sites associated with lamellipodia. Upon binding, GTP-loaded R-Ras converts RIN2 from a Rab5 guanine nucleotide exchange factor (GEF) to an adaptor that first interacts at high affinity with Rab5-GTP to promote the selective endocytosis of ligand-bound/active β1 integrins and then causes the translocation of R-Ras to early endosomes. Here, the R-Ras/RIN2/Rab5 signaling module activates Rac1-dependent cell adhesion via TIAM1, a Rac GEF that localizes on early endosomes and is stimulated by the interaction with both Ras proteins and the vesicular lipid phosphatidylinositol 3-monophosphate. In conclusion, the ability of R-Ras-GTP to convert RIN2 from a GEF to an adaptor that preferentially binds Rab5-GTP allows the triggering of the endocytosis of ECM-bound/active β1 integrins and the ensuing funneling of R-Ras-GTP toward early endosomes to elicit the pro-adhesive and TIAM1-mediated activation of Rac1.     

Anterograde and Retrograde Regulation of Nuclear Genes Encoding Mitochondrial Proteins during Growth,Development, and Stress

Mitochondrial biogenesis and function in plants require the expression of over 1000 nuclear genes encoding mitochondrial proteins （NGEMPs）. The expression of these genes is regulated by tissue-specific, developmental, internal, and external stimuli that result in a dynamic organelle involved in both metabolic and a variety of signaling processes. Although the metabolic and biosynthetic machinery of mitochondria is relatively well understood, the factors that regu- late these processes and the various signaling pathways involved are only beginning to be identified at a molecular level. The molecular components of anterograde （nuclear to mitochondrial） and retrograde （mitochondrial to nuclear） signaling pathways that regulate the expression of NGEMPs interact with chloroplast-, growth-, and stress-signaling pathways in the cell at a variety of levels, with common components involved in transmission and execution of these signals. This positions mitochondria as important hubs for signaling in the cell, not only in direct signaling of mitochondrial function per se, but also in sensing and/or integrating a variety of other internal and external signals. This integrates and optimizes growth with energy metabolism and stress responses, which is required in both photosynthetic and non-photosynthetic cells.     

Structural basis for the interaction between the Tap/NXF1 UBA domain and FG nucleoporins at 1A resolution

The mRNA nuclear export function of Tap/NXF1 requires interactions with nuclear pore proteins (nucleoporins) that contain characteristic Phe-Gly repeats based on FG, GLFG or FxFG cores separated by hydrophilic linkers. FG-nucleoporins bind the two most C-terminal domains of Tap, which have NTF2 and UBA folds, respectively. We used a combination of NMR and X-ray crystallography to define the interaction interface between Tap UBA and FxFG nucleoporins and show that it involves primarily the two aromatic rings of the FxFG core that bind in a hydrophobic surface depression centred on Tap Cys588. NMR evidence indicates that the same depression mediates the binding of GLFG nucleoporins, which we confirmed by demonstrating competition between the two classes of repeat for binding to Tap UBA. Moreover, modification of Cys588 reduced the binding of Tap UBA to both GLFG and FxFG nucleoporins as well as to nuclear envelopes. These data underscore the central role of the conserved FG-nucleoporin repeat cores in binding to Tap UBA and indicate that functional differences between different classes of nucleoporins depend more on their spatial distribution in nuclear pores than on their binding to different sites on Tap UBA.     

Arabidopsis GCN5, HD1, and TAF1/HAF2 interact to regulate histone acetylation required for light-responsive gene expression

We previously showed that Arabidopsis thaliana histone acetyltransferase TAF1/HAF2 is required for the light regulation of growth and gene expression, and we show here that histone acetyltransferase GCN5 and histone deacetylase HD1/HDA19 are also involved in such regulation. Mutation of GCN5 resulted in a long-hypocotyl phenotype and reduced light-inducible gene expression, whereas mutation of HD1 induced opposite effects. The double mutant gcn5 hd1 restored a normal photomorphogenic phenotype. By contrast, the double mutant gcn5 taf1 resulted in further loss of light-regulated gene expression. gcn5 reduced acetylation of histones H3 and H4, mostly on the core promoter regions, whereas hd1 increased acetylation on both core and more upstream promoter regions. GCN5 and TAF1 were both required for H3K9, H3K27, and H4K12 acetylation on the target promoters, but H3K14 acetylation was dependent only on GCN5. Interestingly, gcn5 taf1 had a cumulative effect mainly on H3K9 acetylation. On the other hand, hd1 induced increased acetylation on H3K9, H3K27, H4K5, and H4K8. GCN5 was also shown to be directly associated with the light-responsive promoters. These results suggest that acetylation of specific histone Lys residues, regulated by GCN5, TAF1, and HD1, is required for light-regulated gene expression.     

Interaction of maize Opaque-2 and the transcriptional co-activators GCN5 and ADA2, in the modulation of transcriptional activity

The FtsH proteases, also called AAA proteases, are membrane-bound ATP-dependent metalloproteases. The Arabidopsis genome contains a total of 12 FtsH-like genes. Two of them, AtFtsH4 and AtFtsH11, encode proteins with a high similarity to Yme1p, a subunit of the i-AAA complex in yeast mitochondria. Phylogenetic analysis groups the AtFtsH4, AtFtsH11 and Yme1 proteins together, with AtFtsH4 being the most similar to Yme1. Using immunological method we demonstrate here that AtFtsH4 is an exclusively mitochondrial protein while AtFtsH11 is found in both chloroplasts and mitochondria. AtFtsH4 and AtFtsH11 proteases are integral parts of the inner mitochondrial membrane and expose their catalytic sites towards the intermembrane space, same as yeast i-AAA. Database searches revealed that orthologs of AtFtsH4 and AtFtsH11 are present in both monocotyledonous and dicotyledonous plants. The two plant i-AAA proteases differ significantly in their termini: the FtsH4 proteins have a characteristic alanine stretch at the C-terminal end while FtsH11s have long N-terminal extensions. Blue-native gel electrophoresis revealed that AtFtsH4 and AtFtsH11 form at least two complexes with apparent molecular masses of about 1500 kDa. This finding implies that plants, in contrast to fungi and metazoa, have more than one complex with a topology similar to that of yeast i-AAA.     

Myosin-5, kinesin-1 and myosin-17 cooperate in secretion of fungal chitin synthase

Plant infection by pathogenic fungi requires polarized secretion of enzymes, but little is known about the delivery pathways. Here, we investigate the secretion of cell wall-forming chitin synthases (CHSs) in the corn pathogen Ustilago maydis. We show that peripheral filamentous actin (F-actin) and central microtubules (MTs) form independent tracks for CHSs delivery and both cooperate in cell morphogenesis. The enzyme Mcs1, a CHS that contains a myosin-17 motor domain, is travelling along both MTs and F-actin. This transport is independent of kinesin-3, but mediated by kinesin-1 and myosin-5. Arriving vesicles pause beneath the plasma membrane, but only ~15% of them get exocytosed and the majority is returned to the cell centre by the motor dynein. Successful exocytosis at the cell tip and, to a lesser extent at the lateral parts of the cell requires the motor domain of Mcs1, which captures and tethers the vesicles prior to secretion. Consistently, Mcs1-bound vesicles transiently bind F-actin but show no motility in vitro. Thus, kinesin-1, myosin-5 and dynein mediate bi-directional motility, whereas myosin-17 introduces a symmetry break that allows polarized secretion.     

The role of GABARAPL1/GEC1 in autophagic flux and mitochondrial quality control in MDA-MB-436 breast cancer cells

GABARAPL1/GEC1 is an early estrogen-induced gene which encodes a protein highly conserved from C. elegans to humans. Overexpressed GABARAPL1 interacts with GABA_A or kappa opioid receptors, associates with autophagic vesicles, and inhibits breast cancer cell proliferation. However, the function of endogenous GABARAPL1 has not been extensively studied. We hypothesized that GABARAPL1 is required for maintaining normal autophagic flux, and plays an important role in regulating cellular bioenergetics and metabolism. To test this hypothesis, we knocked down GABARAPL1 expression in the breast cancer MDA-MB-436 cell line by shRNA. Decreased expression of GABARAPL1 activated procancer responses of the MDA-MB-436 cells including increased proliferation, colony formation, and invasion. In addition, cells with decreased expression of GABARAPL1 exhibited attenuated autophagic flux and a decreased number of lysosomes. Moreover, decreased GABARAPL1 expression led to cellular bioenergetic changes including increased basal oxygen consumption rate, increased intracellular ATP, increased total glutathione, and an accumulation of damaged mitochondria. Taken together, our results demonstrate that GABARAPL1 plays an important role in cell proliferation, invasion, and autophagic flux, as well as in mitochondrial homeostasis and cellular metabolic programs.     

AMPA receptor stimulation increases alpha5beta1 integrin surface expression, adhesive function and signaling

Integrin proteins are critical for stabilization of hippocampal long-term potentiation but the mechanisms by which integrin activities are involved in synaptic transmission are not known. The present study tested whether activation of alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionate (AMPA) class glutamate receptors increases surface expression of alpha5beta1 integrin implicated in synaptic potentiation. Surface protein biotinylation assays demonstrated that AMPA treatment of COS7 cells expressing GluR1 homomeric AMPA receptors increased membrane insertion and steady-state surface levels of alpha5 and beta1 subunits. Treated cells exhibited increased adhesion to fibronectin- and anti-alpha5-coated substrates and tyrosine kinase signaling elicited by fibronectin-substrate adhesion, as expected if new surface receptors are functional. Increased surface expression did not occur in calcium-free medium and was blocked by the protein kinase C inhibitor chelerythrine chloride and the exocytosis inhibitor brefeldin A. AMPA treatment similarly increased alpha5 and beta1 surface expression in dissociated neurons and cultured hippocampal slices. In both neuronal preparations AMPA-induced integrin trafficking was blocked by combined antagonism of NMDA receptor and L-type voltage-sensitive calcium channel activities but was not induced by NMDA treatment alone. These results provide the first evidence that glutamate receptor activation increases integrin surface expression and function, and suggest a novel mechanism by which synaptic activity can engage a volley of new integrin signaling in coordination with, and probably involved in, stabilization of synaptic potentiation.     

IF1 reprograms energy metabolism and signals the oncogenic phenotype in cancer

Comment on: Formentini L, et al. Mol Cell 2012; 45:731-42.     

IF1 reprograms energy metabolism and signals the oncogenic phenotype in cancer

Comment on: Formentini L, et al. Mol Cell 2012; 45:731-42.     

Cloning, characterization and expression analysis of nucleotide metabolism-related genes of mycobacteriophage L5

The genomes of mycobacteriophages of the L5 family, which includes the lytic phage D29, contain several genes putatively linked to nucleotide-metabolizing functions. Two such genes, 48 and 50 , encoding thymidylate synthase and ribonucleotide reductase (RNR), respectively, were overexpressed in Escherichia coli and the recombinant proteins were biochemically characterized. It was established that Gp50 was a class II RNR having properties similar to that of the corresponding enzyme from Lactobacillus leichmanni , whereas Gp48 was a flavin-dependent thymidylate synthase (ThyX) that resembled the Paramecium bursaria chlorella virus-1 ThyX enzyme in its properties. That both these proteins play a role in phage development was evident from the observation that they were detectable soon after the lytic phase of growth commenced. Gp48 and 50 were also found to coimmunoprecipitate, which indicates the possible existence of an L5 thymidylate synthase complex. Thymidylate synthase assays revealed that during the intracellular stage of phage growth, a significant decrease in the host thymidylate synthase (ThyA) activity occurred. It appears that synthesis of the viral enzyme (ThyX) is necessary to compensate for this loss in activity. In general, the results suggest that phage-encoded nucleotide metabolism-related functions play an important role in the lytic propagation of L5 and related mycobacteriophages.     

Stereological analysis of mitochondria from brains of temperature acclimated goldfish, Carassius auratus L. (5 and 30°C)

1. 1.|The mitochondrial population in hypothalamic and hypophysial brain tissue from warm (30°C) and cold (5°C) acclimated goldfish (Carassius auralus L.) was analyzed using sterological techniques.

2. 2.|It was revealed that there is a significantly larger volume density (Vv) in the cold acclimated tissue, with no significant difference in either of the surface densities (Svext and Svint) from either of the brain areas.

3. 3.|The hypothalamic brain tissue has a significantly lower specific surface (S/V) in the cold acclimated tissue but there is not a significant difference in this parameter for the hypophysial brain tissue.

4. 4.|The values for these three parameters (Vv, Svext and SVint, and S/V) indicate that mitochondria from acclimated brain tissue undergo shape changes in response to thermal stress.

5. 5.|We suggest that the shape changes may be related to the change in the phospholipid composition of the inner mitochondrial membrane with acclimation temperature.

Pharmacological Differentiation and Characterization of 5-HT1A, 5-HT1B, and 5-HT1C Binding Sites in Rat Frontal Cortex

Drug interactions with 5-HT1 (5-hydroxytryptamine type 1) binding site subtypes were analyzed in rat frontal cortex. 8-Hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT) displays high affinity (Ki 3.3 +/- 1 nM) for 29 +/- 3% of total [3H]5-HT binding in rat frontal cortex and low affinity (Ki 9,300 +/- 1,000) for 71 +/- 4% of the remaining 5-HT1 sites. Therefore, non-5-HT1A binding in rat frontal cortex was defined as specific [3H]5-HT binding observed in the presence of 100 nM 8-OH-DPAT. 5-Methoxy 3-(1,2,3,6-tetrahydro-4-pyridinyl) 1 H indole (RU 24969), 1-(m-trifluoromethylphenyl)piperazine (TFMPP), mianserin, and methysergide produce shallow competition curves of [3H]5-HT binding from non-5-HT1A sites. Addition of 10(-3) M GTP does not increase the apparent Hill slopes of these competition curves. Computer-assisted iterative curve fitting suggests that these drugs can discriminate two distinct subpopulations of non-5-HT1A binding sites, each representing approximately 35% of the total [3H]5-HT binding in the rat frontal cortex. All three 5-HT1 binding site subtypes display nanomolar affinity for 5-HT and 5-methoxytryptamine. A homogeneous population of 5-HT1A sites can be directly labeled using [3H]8-OH-DPAT. These sites display nanomolar affinity for 8-OH-DPAT, WB 4101, RU 24969, 2-(4-[4-(2-pyrimidinyl)-1-piperazinyl] butyl)-1,2-benzisothiazol-3-(2H)one-1, 1-dioxidehydrochloride (TVX Q 7821), 5-methoxydimethyltryptamine, and d-lysergic acid diethylamide. The potencies of RU 24969, TFMPP, and quipazine for [3H]5-HT binding are increased by addition of 100 nM 8-OH-DPAT and 3,000 nM mianserin to the [3H]5-HT binding assay. Moreover, the drugs have apparent Hill slopes near 1 under these conditions. This subpopulation of total [3H]5-HT binding is designated 5-HT1B. By contrast, methysergide and mianserin become more potent inhibitors of residual [3H]5-HT binding to non-5-HT1A sites in the presence of 100 nM 8-OH-DPAT and 10 nM RU 24969. The drug competition curves under these conditions have apparent Hill slopes of near unity and these sites are designated 5-HT1C. Drug competition studies using a series of 24 agents reveals that each 5-HT1 subtype site has a unique pharmacological profile. These results suggest that radioligand studies can be used to differentiate three distinct subpopulations of 5-HT1 binding sites labeled by [3H]5-HT in rat frontal cortex.     

Relationship between moments at the L5/S1 level, hip and knee joint when lifting

A study was performed to determine the influence of load magnitude on the self selected technique of lifting. Specifically, it was hypothesized that with heavier weights a tendency would occur to lift more with the back and less with the legs. Flexion-extension moments at the L5/S1 level, hip and knee joints were calculated for subjects when lifting boxes weighing from 50 to 250 N. Lifts were performed using a freestyle technique at normal speed. The moment profiles (moment plotted vs time) were analyzed kinematically and as a function of the weight lifted. The kinematics of the lift changed as the weight increased. The moment at the L5/S1 level increased with increasing weight, however, the corresponding knee moment decreased. Thus, an inverse relationship was found between the moment at the L5/S1 level and the knee joint moment. An increase in weight lifted was also associated with an increase in the angular velocity at the knee while lifting. Apparently with heavier weights there is a tendency to extend the knees earlier during the lift than with lighter weights, confirming our hypothesis. This explains the reduced knee moment. Our findings lead to the hypothesis that quadriceps muscle strength limits the subjects' ability to lift with their knees flexed.     

Perillyl alcohol and genistein differentially regulate PKB/Akt and 4E-BP1 phosphorylation as well as eIF4E/eIF4G interactions in human tumor cells

Previously we demonstrated that secondary products of plant mevalonate metabolism called isoprenoids attenuate 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA translational efficiency and cause tumor cell death. Here we compared effects of "pure" isoprenoids (perillyl alcohol and gamma-tocotrienol) and a "mixed" isoprenoid-genistein-on the PKB/Akt/mTOR pathway that controls mRNA translation and m(7)GpppX eIF4F cap binding complex formation. Effects were cell- and isoprenoid-specific. Perillyl alcohol and genistein suppressed 4E-BP1(Ser65) phosphorylation in prostate tumor cell lines, DU145 and PC-3, and in Caco2 adenocarcinoma cells. Suppressive effects were similar to or greater than that observed with a PI3 kinase inhibitor or rapamycin, an mTOR inhibitor. 4E-BP1(Thr37) phosphorylation was reduced by perillyl alcohol and genistein in DU145, but not in PC-3. Conversely, perillyl alcohol but not genistein decreased 4E-BP1(Thr37) phosphorylation in Caco2. PKB/Akt activation via Ser473 phosphorylation was enhanced in DU145 by perillyl alcohol and in PC-3 by gamma-tocotrienol, but was suppressed by genistein. Importantly, perillyl alcohol disrupted interactions between eIF4E and eIF4G, key components of eIF4F (m(7)GpppX) cap binding complex. These results demonstrate that "pure" isoprenoids and genistein differentially impact cap-dependent translation in tumor cell lines.     

Leaf anatomy,chloroplast ultrastructure,and cellular localisation of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) and RuBPCO activase in <Emphasis Type="Italic">Amaranthus tricolor</Emphasis> L.

In Amaranthus tricolor the leaf structure included three layers of chlorenchyma on the vascular bundle periphery, namely, mesophyll cells (MSCs) with few chloroplasts, outer larger round bundle sheath cells (BSCs) with many chloroplasts in a centripetal position, and inner smaller BSCs with few chloroplasts around the vascular bundle cells. The ultra-thin sections showed that BSCs had abundant organelles, namely many large and round mitochondria with well-developed cristae in the cytoplasm. The chloroplasts in the BSCs were lens-like bodies, which seemed to be oval on cross sections. Granal and intergranal thylakoids were usually distinguished. Grana were stacked in parallel with prevailing plane of thylakoid lamellae. The chloroplasts in the MSCs appeared smaller than those in the BSCs and contained less stacked thylakoids but abundant peripheral reticulum. The ultra-thin sections of immunogold-labelled anti-ribulose-1,5-bisphosphate carboxylase/oxygenase (anti-RuBPCO) exhibited high density of RuBPCO labelling in the stroma region of chloroplasts of the BSCs. Some anti-RuBPCO immunogold particles were observed in the stromal region of MSCs chloroplasts. The anti-activase (A) immunogold-labelling indicated that RuBPCOA was mainly distributed in the stroma region of both BSCs and MSCs chloroplasts. From the chloroplast ultrastructure and localisation of RuBPCO and RuBPCOA we deduced that the photosynthetic carbon reduction cycle and the formation of assimilatory power function in both MSC and BSC chloroplasts of A. tricolor.