Switching the sorting mode of membrane proteins from cotranslational endoplasmic reticulum targeting to posttranslational mitochondrial import

Hydrophobic membrane proteins are cotranslationally targeted to the endoplasmic reticulum (ER) membrane, mediated by hydrophobic signal sequence. Mitochondrial membrane proteins escape this mechanism despite their hydrophobic character. We examined sorting of membrane proteins into the mitochondria, by using mitochondrial ATP-binding cassette (ABC) transporter isoform (ABC-me). In the absence of 135-residue N-terminal hydrophilic segment (N135), the membrane domain was integrated into the ER membrane in COS7 cells. Other sequences that were sufficient to import soluble protein into mitochondria could not import the membrane domain. N135 imports other membrane proteins into mitochondria. N135 prevents cotranslational targeting of the membrane domain to ER and in turn achieves posttranslational import into mitochondria. In a cell-free system, N135 suppresses targeting to the ER membranes, although it does not affect recognition of hydrophobic segments by signal recognition particle. We conclude that the N135 segment blocks the ER targeting of membrane proteins even in the absence of mitochondria and switches the sorting mode from cotranslational ER integration to posttranslational mitochondrial import.     

Glial Fatty Acid-Binding Protein 7 (FABP7) Regulates Neuronal Leptin Sensitivity in the Hypothalamic Arcuate Nucleus

The hypothalamus is involved in the regulation of food intake and energy homeostasis. The arcuate nucleus (ARC) and median eminence (ME) are the primary hypothalamic sites that sense leptin and nutrients in the blood, thereby mediating food intake. Recently, studies demonstrating a role for non-neuronal cell types, including astrocytes and tanycytes, in these regulatory processes have begun to emerge. However, the molecular mechanisms involved in these activities remain largely unknown. In this study, we examined in detail the localization of fatty acid-binding protein 7 (FABP7) in the hypothalamic ARC and sought to determine its role in the hypothalamus. We performed a phenotypic analysis of diet-induced FABP7 knockout (KO) obese mice and of FABP7 KO mice treated with a single leptin injection. Immunohistochemistry revealed that FABP7⁺ cells are NG2⁺ or GFAP⁺ in the ARC and ME. In mice fed a high-fat diet, weight gain and food intake were lower in FABP7 KO mice than in wild-type (WT) mice. FABP7 KO mice also had lower food intake and weight gain after a single injection of leptin, and we consistently confirmed that the number of pSTAT3⁺ cells in the ARC indicated that the leptin-induced activation of neurons was significantly more frequent in FABP7 KO mice than in WT mice. In FABP7 KO mice-derived primary astrocyte cultures, the level of ERK phosphorylation was lower after leptin treatment. Collectively, these results indicate that in hypothalamic astrocytes, FABP7 might be involved in sensing neuronal leptin via glia-mediated mechanisms and plays a pivotal role in controlling systemic energy homeostasis.     

Novel green fluorescent protein-based ratiometric indicators for monitoring pH in defined intracellular microdomains.

To measure pH in defined intracellular microdomains of living cells, we developed ratiometric indicators based on fusing in tandem two green fluorescent protein (GFP) variants having different pH sensitivities. The indicators function in a single-excitation/dual-emission mode involving fluorescence resonance energy transfer, as well as in a dual-excitation/single-emission mode. The fluorescence ratio from GFpH and YFpH showed pH dependency and pK(a) values were 6.1 and 6.8, respectively. Using these indicators expressed in cultured cells, we measured and visualized pH changes in the cytosol and nucleus. Furthermore, by tethering the indicator to a membrane protein (the alpha(1B) adrenergic receptor), we visualized the pH in the vicinity of the protein during internalization caused by endocytosis after agonist stimulation. These novel probes will serve as a useful tool for monitoring pH in the defined organelle and in the microenvironment of a target protein, to analyze cellular function.     

Requirement for C3G-dependent Rap1 activation for cell adhesion and embryogenesis.

C3G is a guanine nucleotide exchange factor (GEF) for Rap1, and is activated via Crk adaptor protein. To understand the physiological role of C3G, we generated C3G knockout mice. C3G(-/-) homozygous mice died before embryonic day 7.5. The lethality was rescued by the expression of the human C3G transgene, which could be excised upon the expression of Cre recombinase. From the embryo of this mouse, we prepared fibroblast cell lines, MEF-hC3G. Expression of Cre abolished the expression of C3G in MEF-hC3G and inhibited cell adhesion-induced activation of Rap1. The Cre-expressing MEF-hC3G showed impaired cell adhesion, delayed cell spreading and accelerated cell migration. The accelerated cell migration was suppressed by the expression of active Rap1, Rap2 and R-Ras. Expression of Epac and CalDAG-GEFI, GEFs for Rap1, also suppressed the accelerated migration of the C3G-deficient cells. This observation indicated that Rap1 activation was sufficient to complement the C3G deficiency. In conclusion, C3G-dependent activation of Rap1 is required for adhesion and spreading of embryonic fibroblasts and for the early embryogenesis of the mouse.     

Physiological relaxation induced by horticultural activity: transplanting work using flowering plants

Background

Despite increasing attention and a growing volume of research data, little physiological evidence is available on the benefits of horticultural activity and the different effects on individuals. Therefore, the aim of the present study was to investigate the physiological effects of horticultural activity and to examine how differences in personality alter these effects.

Results

The effects of transplanting real flowers (horticultural activity) and handling artificial flowers (control activity) on human physiological activity were compared. On the first day, eight participants engaged in horticultural activity and another eight in the control activity. On the second day, participants switched roles. Participants’ physiological conditions during each activity were assessed by measuring the heart rate and heart rate variability (HRV). Psychological responses, which were measured using a semantic differential rating scale, showed that the horticultural activity promoted comfortable, soothed, and natural feelings, compared to the control activity. Analysis of physiological responses using two-way repeated measures analysis of variance (ANOVA) revealed that sympathetic nervous activity significantly decreased in the late time period (11 to 15 minutes) of horticultural activity only in the type A group.

Conclusions

This study supports the fact that the horticultural activity can enhance psychological and physiological relaxation effects, although these physiological effects can differ among individuals with different personalities.     

Characterization of a Novel β-Glucosidase from a Compost Microbial Metagenome with Strong Transglycosylation Activity

The β-glucosidase encoded by the td2f2 gene was isolated from a compost microbial metagenomic library by functional screening. The protein was identified to be a member of the glycoside hydrolase family 1 and was overexpressed in Escherichia coli, purified, and biochemically characterized. The recombinant β-glucosidase, Td2F2, exhibited enzymatic activity with β-glycosidic substrates, with preferences for glucose, fucose, and galactose. Hydrolysis occurred at the nonreducing end and in an exo manner. The order of catalytic efficiency for glucodisaccharides and cellooligosaccharides was sophorose > cellotetraose > cellotriose > laminaribiose > cellobiose > cellopentaose > gentiobiose, respectively. Intriguingly, the p-nitrophenyl-β-d-glucopyranoside hydrolysis activity of Td2F2 was activated by various monosaccharides and sugar alcohols. At a d-glucose concentration of 1000 mm, enzyme activity was 6.7-fold higher than that observed in the absence of d-glucose. With 31.3 mm d-glucose, Td2F2 catalyzed transglycosylation to generate sophorose, laminaribiose, cellobiose, and gentiobiose. Transglycosylation products were detected under all activated conditions, suggesting that the activity enhancement induced by monosaccharides and sugar alcohols may be due to the transglycosylation activity of the enzyme. These results show that Td2F2 obtained from a compost microbial metagenome may be a potent candidate for industrial applications.     

ACTN1 Mutations Cause Congenital Macrothrombocytopenia

Congenital macrothrombocytopenia (CMTP) is a heterogeneous group of rare platelet disorders characterized by a congenital reduction of platelet counts and abnormally large platelets, for which CMTP-causing mutations are only found in approximately half the cases. We herein performed whole-exome sequencing and targeted Sanger sequencing to identify mutations that cause CMTP, in which a dominant mode of transmission had been suspected but for which no known responsible mutations have been documented. In 13 Japanese CMTP-affected pedigrees, we identified six (46%) affected by ACTN1 variants cosegregating with CMTP. In the entire cohort, ACNT1 variants accounted for 5.5% of the dominant forms of CMTP cases and represented the fourth most common cause in Japanese individuals. Individuals with ACTN1 variants presented with moderate macrothrombocytopenia with anisocytosis but were either asymptomatic or had only a modest bleeding tendency. ACTN1 encodes α-actinin-1, a member of the actin-crosslinking protein superfamily that participates in the organization of the cytoskeleton. In vitro transfection experiments in Chinese hamster ovary cells demonstrated that altered α-actinin-1 disrupted the normal actin-based cytoskeletal structure. Moreover, transduction of mouse fetal liver-derived megakaryocytes with disease-associated ACTN1 variants caused a disorganized actin-based cytoskeleton in megakaryocytes, resulting in the production of abnormally large proplatelet tips, which were reduced in number. Our findings provide an insight into the pathogenesis of CMTP.     

Extracellular syntaxin4 triggers the differentiation program in teratocarcinoma F9 cells that impacts cell adhesion properties

The proteins in the syntaxin family are known to mediate fusion of cytoplasmic vesicles to the target membrane, yet subpopulations of certain syntaxins, including syntaxin4, translocate across the cell membrane in response to external stimuli. Here, we show that extracellularly presented syntaxin4 impacts cell behavior and differentiation in teratocarcinoma F9 cells. While undifferentiated F9 cells extruded a small subpopulation of extracellular syntaxin4 at the lateral cell membrane, the induction of differentiation with all-trans retinoic acid (RA) abolished this localized expression pattern. We found that the cells that were stimulated in a non-directional fashion by extracellular syntaxin4 displayed a flattened shape and retained a substrate-bound morphology even under a long-term, serum-starved cultivation. Such a cellular response was also elicited by a circular peptide composed of the potential functional core of syntaxin4 (AIEPQK; amino acid residues 103～108) (ST4n1). While the proliferation and metabolism were not affected in these cells, cell–cell interaction became weakened and the expression of vinculin, a regulator of both intercellular and cell-substrate adhesion molecules, was altered. We also found that the expressions of several differentiation markers were up-regulated in cells stimulated with extracellular syntaxin4 and that syntaxin3, another family member, was most prominent. Intriguingly, forced expression of syntaxin3 induced the spread morphology in F9 cells, indicating that syntaxin3 partly mediates the function of extracellular syntaxin4. These results demonstrate the involvement of a non-directional stimulation of extracellular syntaxin4 in the functional and morphological differentiation of F9 cells.