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1.  Sex-Specific Posttranslational Regulation of the Gamete Fusogen GCS1 in the Isogamous Volvocine Alga Gonium pectorale 
Eukaryotic Cell  2014;13(5):648-656.
Male and female, generally defined based on differences in gamete size and motility, likely have multiple independent origins, appearing to have evolved from isogamous organisms in various eukaryotic lineages. Recent studies of the gamete fusogen GCS1/HAP2 indicate that this protein is deeply conserved across eukaryotes, and its exclusive and/or functional expression generally resides in males or in male homologues. However, little is known regarding the conserved or primitive molecular traits of males and females within eukaryotes. Here, using morphologically indistinguishable isogametes of the colonial volvocine Gonium pectorale, we demonstrated that GCS1 is differently regulated between the sexes. G. pectorale GCS1 molecules in one sex (homologous to male) are transported from the gamete cytoplasm to the protruded fusion site, whereas those of the other sex (females) are quickly degraded within the cytoplasm upon gamete activation. This molecular trait difference might be conserved across various eukaryotic lineages and may represent male and female prototypes originating from a common eukaryotic ancestor.
PMCID: PMC4060472  PMID: 24632243
2.  Vesicular trafficking in characean green algae and the possible involvement of a VAMP72-family protein 
Plant Signaling & Behavior  2014;9:e28466.
The RAB5 GTPase ARA6 of Arabidopsis thaliana is known to be involved in endosomal trafficking by targeting vesicles to the plasma membrane. During this process AtARA6 is working in close relationship with the SNARE protein VAMP727 (vesicle associated membrane protein 727). Recently, ARA6 of the characean green algae Chara australis (CaARA6) was shown to have properties similar to AtARA6, pointing to similar trafficking pathways. In order to gain further insight into the vesicle trafficking machinery of Characeae, C. australis was analyzed for homologous proteins of the VAMP72-family. A CaVAMP72 protein was detected and classified by protein sequence alignment and phylogenetic analyses.
PMCID: PMC4091427  PMID: 24614164
ARA6; RAB5 GTPase; Characeae; Chara australis; endosomal trafficking; plasma membrane; SNARE protein; vesicle associated membrane protein; VAMP
3.  Complete response of sunitinib therapy for renal cell cancer recurrence in the native kidney after renal transplantation: a case report 
BMC Research Notes  2014;7(1):526.
No case report has yet shown that sunitinib therapy for the postoperative recurrence of renal cancer in a native kidney after renal transplantation can achieve complete response (CR).
Case presentation
A tumor was detected in the right native kidney of a 35-year-old Japanese male 10 years after renal transplantation. A tumor thrombus that reached the atrium was detected, which suggested cT3cN0M0. Because of the risk of perioperative complications, preoperative therapy with sunitinib was selected and 8 courses were administered. The size of the primary tumor was reduced by 33%, while that of the tumor thrombus was decreased by 39.5%.
Right nephrectomy and removal of the tumor thrombus were then performed. Contrast-enhanced computed tomography (CT) four months after surgery suggested local relapse. Sunitinib was administered for 9 months, which led to complete response (CR).
This study presented the case of sunitinib therapy for renal cancer in the native kidney after renal transplantation. The therapeutic efficacy and safety for such cases should be discussed.
PMCID: PMC4138390  PMID: 25124932
Complete response; Native kidney; Neoadjuvant therapy; Presurgical therapy; Renal cell cancer; Renal transplantation; Sunitinib; Tumor thrombus
4.  ARA7(Q69L) expression in transgenic Arabidopsis cells induces the formation of enlarged multivesicular bodies 
Journal of Experimental Botany  2013;64(10):2817-2829.
Arabidopsis thaliana ARA7 (AtRabF2b), a member of the plant Rab5 small GTPases functioning in the vacuolar transport pathway, localizes to pre-vacuolar compartments (PVCs), known as multivesicular bodies (MVBs) in plant cells. Overexpression of the constitutively active GTP-bound mutant of ARA7, ARA7(Q69L), induces the formation of large ring-like structures (1–2 µm in diameter). To better understand the biology of these ARA7(Q69L)-induced ring-like structures, transgenic Arabidopsis cell lines expressing ARA7(Q69L) tagged with green fluorescent protein (GFP) under the control of a heat shock-inducible promoter were generated. In these transgenic cells, robust ring-like structures were formed after 4 h of heat shock induction. Transient co-expression, confocal imaging, and immunogold electron microscopy (immunogold-EM) experiments demonstrated that these GFP–ARA7(Q69L)-labelled ring-like structures were distinct from the Golgi apparatus and trans-Golgi network, but were labelled with an antibody against an MVB marker protein. In addition, live cell imaging and detailed EM analysis showed that the GFP–ARA7(Q69L)-induced spherical structures originated from the homotypic fusion of MVBs. In summary, it was demonstrated that GFP–ARA7(Q69L) expression is an efficient tool for studying PVC/MVB-mediated protein trafficking and vacuolar degradation in plant cells.
PMCID: PMC3697957  PMID: 23682115
ARA7(Q69L); homotypic fusion; MVB enlargement; multivesicular body; pre-vacuolar compartment; transgenic Arabidopsis cells.
5.  New insights into the role of Arabidopsis RABA1 GTPases in salinity stress tolerance 
Plant Signaling & Behavior  2013;8(9):e25377.
RAB11 GTPases, widely conserved members of RAB small GTPases, have evolved in a unique way in plants; plant RAB11 has notable diversity compared with animals and yeast. Recently, we have shown that members of RABA1, a subgroup in Arabidopsis RAB11 group, are required for salinity stress tolerance. To obtain a clue to understand its underlying mechanism, here we investigate whether RABA1 regulates sodium transport across the plasma membrane and accumulation in the vacuole. The results indicate that the raba1 quadruple mutant is not defective in the import and intracellular distribution of sodium, implying that RABA1 members are involved in a more indirect way in the responses to salinity stress.
PMCID: PMC4002589  PMID: 23803751
RAB GTPase; RABA1; salinity stress
6.  A Unique HEAT Repeat-Containing Protein SHOOT GRAVITROPISM6 is Involved in Vacuolar Membrane Dynamics in Gravity-Sensing Cells of Arabidopsis Inflorescence Stem 
Plant and Cell Physiology  2014;55(4):811-822.
Plant vacuoles play critical roles in development, growth and stress responses. In mature cells, vacuolar membranes (VMs) display several types of structures, which are formed by invagination and folding of VMs into the lumenal side and can gradually move and change shape. Although such VM structures are observed in a broad range of tissue types and plant species, the molecular mechanism underlying their formation and maintenance remains unclear. Here, we report that a novel HEAT-repeat protein, SHOOT GRAVITROPISM6 (SGR6), of Arabidopsis is involved in the control of morphological changes and dynamics of VM structures in endodermal cells, which are the gravity-sensing cells in shoots. SGR6 is a membrane-associated protein that is mainly localized to the VM in stem endodermal cells. The sgr6 mutant stem exhibits a reduced gravitropic response. Higher plants utilize amyloplast sedimentation as a means to sense gravity direction. Amyloplasts are surrounded by VMs in Arabidopsis endodermal cells, and the flexible and dynamic structure of VMs is important for amyloplast sedimentation. We demonstrated that such dynamic features of VMs are gradually lost in sgr6 endodermal cells during a 30 min observation period. Histological analysis revealed that amyloplast sedimentation was impaired in sgr6. Detailed live-cell imaging analyses revealed that the VM structures in sgr6 had severe defects in morphological changes and dynamics. Our results suggest that SGR6 is a novel protein involved in the formation and/or maintenance of invaginated VM structures in gravity-sensing cells.
PMCID: PMC3982123  PMID: 24486761
Arabidopsis; Gravitropism; HEAT-repeat protein; Vacuolar membrane
7.  Evaluating positional accuracy using megavoltage cone-beam computed tomography for IMRT with head-and-neck cancer 
Journal of Radiation Research  2014;55(3):568-574.
Accurate dose delivery is essential for the success of intensity-modulated radiation therapy (IMRT) for patients with head-and-neck (HN) cancer. Reproducibility of IMRT dose delivery to HN regions can be critically influenced by treatment-related changes in body contours. Moreover, some set-up margins may not be adaptable to positional uncertainties of HN structures at every treatment. To obtain evidence for appropriate set-up margins in various head and neck areas, we prospectively evaluated positional deviation (δ values) of four bony landmarks (i.e. the clivus and occipital protuberance for the head region, and the mental protuberance and C5 for the neck region) using megavoltage cone-beam computed tomography during a treatment course. Over 800 δ values were analyzed in each translational direction. Positional uncertainties for HN cancer patients undergoing IMRT were evaluated relative to the body mass index. Low positional accuracy was observed for the neck region compared with the head region. For the head region, most of the δ was distributed within ±5 mm, and use of the current set-up margin was appropriate. However, the δ values for the neck region were within ±8 mm. Especially for overweight patients, a few millimeters needed to be added to give an adequate set-up margin. For accurate dose delivery to targets and to avoid excess exposure to normal tissues, we recommend that the positional verification process be performed before every treatment.
PMCID: PMC4014166  PMID: 24449713
positional accuracy; body mass index; head and neck; IMRT; MV-CBCT
8.  Molecular and biochemical analysis of the first ARA6 homologue, a RAB5 GTPase, from green algae 
Journal of Experimental Botany  2013;64(18):5553-5568.
RAB5 GTPases are important regulators of endosomal membrane traffic in yeast, plants, and animals. A specific subgroup of this family, the ARA6 group, has been described in land plants including bryophytes, lycophytes, and flowering plants. Here, we report on the isolation of an ARA6 homologue in a green alga. CaARA6 (CaRABF1) from Chara australis, a member of the Characeae that is a close relative of land plants, encodes a polypeptide of 237 aa with a calculated molecular mass of 25.4kDa, which is highly similar to ARA6 members from Arabidopsis thaliana and other land plants and has GTPase activity. When expressed in Nicotiana benthamiana leaf epidermal cells, fluorescently tagged CaARA6 labelled organelles with diameters between 0.2 and 1.2 µm, which co-localized with fluorescently tagged AtARA6 known to be present on multivesicular endosomes. Mutations in the membrane-anchoring and GTP-binding sites altered the localization of CaARA6 comparable to that of A. thaliana ARA6 (RABF1). In characean internodal cells, confocal immunofluorescence and immunogold electron microscopy with antibodies against AtARA6 and CaARA6 revealed ARA6 epitopes not only at multivesicular endosomes but also at the plasma membrane, including convoluted domains (charasomes), and at the trans-Golgi network. Our findings demonstrate that ARA6-like proteins have a more ancient origin than previously thought. They indicate further that ARA6-like proteins could have different functions in spite of the high similarity between characean algae and flowering plants.
PMCID: PMC3871812  PMID: 24127512
ARA6; Chara australis; endosomal trafficking; multivesicular endosome; plant-specific RAB5 GTPase; plasma membrane; trans-Golgi network.
9.  The physiological role of SYP4 in the salinity and osmotic stress tolerances 
Plant Signaling & Behavior  2012;7(9):1118-1120.
The trans-Golgi network (TGN) contains multiple sorting domains and acts as the compartment for cargo sorting. Recent evidence indicates that the TGN also functions as an early endosome, the first compartment in the endocytic pathway in plants. The SYP4 group, plant Qa-SNAREs localized on the TGN, regulates both secretory and vacuolar transport pathways. Consistent with a secretory role, SYP4 proteins are required for extracellular resistance to fungal pathogens. However, the physiological role of SYP4 in abiotic stress remains unknown. Here, we report the phenotypes of a syp4-mutant in regard to salinity and osmotic response, and describe the physiological roles of the SYP4 group in the abiotic stress response.
PMCID: PMC3489641  PMID: 22899062
SNARE; SYP4; TGN; salinity stress
10.  cis-Golgi proteins accumulate near the ER exit sites and act as the scaffold for Golgi regeneration after brefeldin A treatment in tobacco BY-2 cells 
Molecular Biology of the Cell  2012;23(16):3203-3214.
Particular cis-Golgi proteins accumulate in novel punctate structures close to ERES by BFA treatment in tobacco BY-2 cells. These structures reassemble first to form cis-Golgi after BFA removal, and the Golgi stacks regenerate in the cis-to-trans order. This indicates that the punctate structures act as the scaffold for Golgi regeneration.
The Golgi apparatus forms stacks of cisternae in many eukaryotic cells. However, little is known about how such a stacked structure is formed and maintained. To address this question, plant cells provide a system suitable for live-imaging approaches because individual Golgi stacks are well separated in the cytoplasm. We established tobacco BY-2 cell lines expressing multiple Golgi markers tagged by different fluorescent proteins and observed their responses to brefeldin A (BFA) treatment and BFA removal. BFA treatment disrupted cis, medial, and trans cisternae but caused distinct relocalization patterns depending on the proteins examined. Medial- and trans-Golgi proteins, as well as one cis-Golgi protein, were absorbed into the endoplasmic reticulum (ER), but two other cis-Golgi proteins formed small punctate structures. After BFA removal, these puncta coalesced first, and then the Golgi stacks regenerated from them in the cis-to-trans order. We suggest that these structures have a property similar to the ER-Golgi intermediate compartment and function as the scaffold of Golgi regeneration.
PMCID: PMC3418314  PMID: 22740633
11.  Significance of serum tumor necrosis factor-related apoptosis-inducing ligand as a prognostic biomarker for renal cell carcinoma 
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is endogenously expressed in immune cells and contributes to immunosurveillance for cancer. TRAIL induces apoptosis preferentially in various cancer cells, including renal cell carcinoma (RCC) cells. In this study, the serum TRAIL level was examined using an enzyme-linked immunosorbent assay in 52 healthy controls and in 84 RCC patients prior to surgery and its significance as a biomarker was evaluated. The median serum TRAIL level was lower in RCC patients compared to the healthy controls (55.9 vs. 103.1 pg/ml; P=0.019). RCC with lymph node metastasis (N1–2), distant metastasis (M1), stage III-IV, or microscopic venous invasion was associated with decreased serum TRAIL levels (P=0.032, 0.067, 0.020 and 0.011). When comparing serum TRAIL levels in the same RCC patients prior and subsequent to surgery (n=11), the levels were significantly higher after surgery (P=0.031). The cause-specific survival rate was significantly higher in RCC patients with high serum TRAIL levels compared to those with low serum TRAIL levels (P=0.0451). TRAIL was estimated to contribute 64 and 13% of the lymphocyte-mediated cytotoxicity against human RCC ACHN and Caki-1 cells, respectively. These data suggest that the serum TRAIL level may be useful as a prognostic biomarker in RCC patients.
PMCID: PMC3956254  PMID: 24649125
tumor necrosis factor-related apoptosis-inducing ligand; serum; biomarker; renal cell carcinoma
12.  Flowering Time Modulation by a Vacuolar SNARE via FLOWERING LOCUS C in Arabidopsis thaliana 
PLoS ONE  2012;7(7):e42239.
The transition of plant growth from vegetative to reproductive phases is one of the most important and dramatic events during the plant life cycle. In Arabidopsis thaliana, flowering promotion involves at least four genetically defined regulatory pathways, including the photoperiod-dependent, vernalization-dependent, gibberellin-dependent, and autonomous promotion pathways. Among these regulatory pathways, the vernalization-dependent and autonomous pathways are integrated by the expression of FLOWERING LOCUS C (FLC), a negative regulator of flowering; however, the upstream regulation of this locus has not been fully understood. The SYP22 gene encodes a vacuolar SNARE protein that acts in vacuolar and endocytic trafficking pathways. Loss of SYP22 function was reported to lead to late flowering in A. thaliana plants, but the mechanism has remained completely unknown. In this study, we demonstrated that the late flowering phenotype of syp22 was due to elevated expression of FLC caused by impairment of the autonomous pathway. In addition, we investigated the DOC1/BIG pathway, which is also suggested to regulate vacuolar/endosomal trafficking. We found that elevated levels of FLC transcripts accumulated in the doc1-1 mutant, and that syp22 phenotypes were exaggerated with a double syp22 doc1-1 mutation. We further demonstrated that the elevated expression of FLC was suppressed by ara6-1, a mutation in the gene encoding plant-unique Rab GTPase involved in endosomal trafficking. Our results indicated that vacuolar and/or endocytic trafficking is involved in the FLC regulation of flowering time in A. thaliana.
PMCID: PMC3407077  PMID: 22848750
13.  Qualitative difference between “bulb” membranes and other vacuolar membranes 
Plant Signaling & Behavior  2011;6(12):1914-1917.
“Bulb” is a mobile and complex structure appearing in vacuolar membrane of plant cell. We recently reported new fluorescent marker lines for bulbs and bulb-less mutants. We tried multicolor visualization of vacuolar membrane to show distinct segregation of bulb-positive protein (γTIP or AtVAM3) and bulb-negative protein (AtRab75). Unexpectedly, GFP-AtRab75 resulted to localize in bulb under the condition of co-expression with TagRFP-AtVAM3. The signal intensities of GFP-AtRab75 and TagRFP-AtVAM3 were quantified and compared. The result indicates that TagRFP-AtVAM3 is concentrated in bulb than GFP-AtRab75.
PMCID: PMC3337177  PMID: 22105033
AtRab75; AtVam3; plant growth; Rab-GTPase; SNARE; vacuolar membranes; “bulb”
14.  Statistical organelle dissection of Arabidopsis guard cells using image database LIPS 
Scientific Reports  2012;2:405.
To comprehensively grasp cell biological events in plant stomatal movement, we have captured microscopic images of guard cells with various organelles markers. The 28,530 serial optical sections of 930 pairs of Arabidopsis guard cells have been released as a new image database, named Live Images of Plant Stomata (LIPS). We visualized the average organellar distributions in guard cells using probabilistic mapping and image clustering techniques. The results indicated that actin microfilaments and endoplasmic reticulum (ER) are mainly localized to the dorsal side and connection regions of guard cells. Subtractive images of open and closed stomata showed distribution changes in intracellular structures, including the ER, during stomatal movement. Time-lapse imaging showed that similar ER distribution changes occurred during stomatal opening induced by light irradiation or femtosecond laser shots on neighboring epidermal cells, indicating that our image analysis approach has identified a novel ER relocation in stomatal opening.
PMCID: PMC3349934  PMID: 22582142
15.  Endosomal trafficking pathway regulated by ARA6, a RAB5 GTPase unique to plants 
Small GTPases  2012;3(1):23-27.
Lineage-specific expansion, followed by functional diversification of key components that act in membrane trafficking, is thought to contribute to lineage-specific diversification of organelles and membrane trafficking pathways. Indeed, recent comparative genomic studies have indicated that specific expansion of RAB and SNARE molecules occurred independently in various eukaryotic lineages over evolutionary history. However, experimental verification of this notion is difficult, because detailed functional analyses of RAB and SNARE proteins uniquely acquired by specific lineages are essential to understanding how new membrane trafficking pathways may have evolved. Recently, we found that a plant-specific RAB GTPase, ARA6, and a plant-unique R-SNARE, VAMP727, mediate a trafficking pathway from endosomes to the plasma membrane in Arabidopsis thaliana. Although a similar endosomal trafficking pathway was also reported in animals, the molecular machineries acting in these trafficking systems differ between animals and plants. Thus, trafficking pathways from endosomes to the plasma membrane appear to have been acquired independently in animal and plant systems. We further demonstrated that the ARA6-mediated trafficking pathway is required for the proper salt-stress response of A. thaliana. These results indicate that acquisition of a new membrane trafficking pathway may be associated with maximization of the fitness of each organism in a lineage-specific manner.
PMCID: PMC3398913  PMID: 22710734
ARA6; Arabidopsis thaliana; Rab5; SNARE; endosome; stress response
16.  Conserved and Plant-Unique Mechanisms Regulating Plant Post-Golgi Traffic 
Membrane traffic plays crucial roles in diverse aspects of cellular and organelle functions in eukaryotic cells. Molecular machineries regulating each step of membrane traffic including the formation, tethering, and fusion of membrane carriers are largely conserved among various organisms, which suggests that the framework of membrane traffic is commonly shared among eukaryotic lineages. However, in addition to the common components, each organism has also acquired lineage-specific regulatory molecules that may be associated with the lineage-specific diversification of membrane trafficking events. In plants, comparative genomic analyses also indicate that some key machineries of membrane traffic are significantly and specifically diversified. In this review, we summarize recent progress regarding plant-unique regulatory mechanisms for membrane traffic, with a special focus on vesicle formation and fusion components in the post-Golgi trafficking pathway.
PMCID: PMC3428585  PMID: 22973281
coat protein complex; dynamin-related protein; membrane trafficking; Rab GTPase; tether; SNARE
17.  Generation of cell polarity in plants links endocytosis, auxin distribution and cell fate decisions 
Nature  2008;456(7224):962-966.
Dynamically polarized membrane proteins define different cell boundaries and have an important role in intercellular communication—a vital feature of multicellular development. Efflux carriers for the signalling molecule auxin from the PIN family1 are landmarks of cell polarity in plants and have a crucial involvement in auxin distribution-dependent development including embryo patterning, organogenesis and tropisms2–7. Polar PIN localization determines the direction of intercellular auxin flow8, yet the mechanisms generating PIN polarity remain unclear. Here we identify an endocytosis-dependent mechanism of PIN polarity generation and analyse its developmental implications. Real-time PIN tracking showed that after synthesis, PINs are initially delivered to the plasma membrane in a non-polar manner and their polarity is established by subsequent endocytic recycling. Interference with PIN endocytosis either by auxin or by manipulation of the Arabidopsis Rab5 GTPase pathway prevents PIN polarization. Failure of PIN polarization transiently alters asymmetric auxin distribution during embryogenesis and increases the local auxin response in apical embryo regions. This results in ectopic expression of auxin pathway-associated root-forming master regulators in embryonic leaves and promotes homeotic transformation of leaves to roots. Our results indicate a two-step mechanism for the generation of PIN polar localization and the essential role of endocytosis in this process. It also highlights the link between endocytosis-dependent polarity of individual cells and auxin distribution-dependent cell fate establishment for multicellular patterning.
PMCID: PMC2692841  PMID: 18953331
18.  Endocytosis restricts Arabidopsis KNOLLE syntaxin to the cell division plane during late cytokinesis 
The EMBO Journal  2009;29(3):546-558.
Cytokinesis represents the final stage of eukaryotic cell division during which the cytoplasm becomes partitioned between daughter cells. The process differs to some extent between animal and plant cells, but proteins of the syntaxin family mediate membrane fusion in the plane of cell division in diverse organisms. How syntaxin localization is kept in check remains elusive. Here, we report that localization of the Arabidopsis KNOLLE syntaxin in the plane of cell division is maintained by sterol-dependent endocytosis involving a clathrin- and DYNAMIN-RELATED PROTEIN1A-dependent mechanism. On genetic or pharmacological interference with endocytosis, KNOLLE mis-localizes to lateral plasma membranes after cell-plate fusion. Fluorescence-loss-in-photo-bleaching and fluorescence-recovery-after-photo-bleaching experiments reveal lateral diffusion of GFP-KNOLLE from the plane of division to lateral membranes. In an endocytosis-defective sterol biosynthesis mutant displaying lateral KNOLLE diffusion, KNOLLE secretory trafficking remains unaffected. Thus, restriction of lateral diffusion by endocytosis may serve to maintain specificity of syntaxin localization during late cytokinesis.
PMCID: PMC2789941  PMID: 19959995
Arabidopsis; cytokinesis; endocytosis; KNOLLE syntaxin; sterols
19.  Corepressive Action of CBP on Androgen Receptor Transactivation in Pericentric Heterochromatin in a Drosophila Experimental Model System▿ †  
Molecular and Cellular Biology  2008;29(4):1017-1034.
Ligand-bound nuclear receptors (NR) activate transcription of the target genes. This activation is coupled with histone modifications and chromatin remodeling through the function of various coregulators. However, the nature of the dependence of a NR coregulator action on the presence of the chromatin environment at the target genes is unclear. To address this issue, we have developed a modified position effect variegation experimental model system that includes an androgen-dependent reporter transgene inserted into either a pericentric heterochromatin region or a euchromatic region of Drosophila chromosome. Human androgen receptor (AR) and its constitutively active truncation mutant (AR AF-1) were transcriptionally functional in both chromosomal regions. Predictably, the level of AR-induced transactivation was lower in the pericentric heterochromatin. In genetic screening for AR AF-1 coregulators, Drosophila CREB binding protein (dCBP) was found to corepress AR transactivation at the pericentric region whereas it led to coactivation in the euchromatic area. Mutations of Sir2 acetylation sites or deletion of the CBP acetyltransferase domain abrogated dCBP corepressive action for AR at heterochromatic areas in vivo. Such a CBP corepressor function for AR was observed in the transcriptionally silent promoter of an AR target gene in cultured mammalian cells. Thus, our findings suggest that the action of NR coregulators may depend on the state of chromatin at the target loci.
PMCID: PMC2643800  PMID: 19075001
20.  Application of Lifeact Reveals F-Actin Dynamics in Arabidopsis thaliana and the Liverwort, Marchantia polymorpha 
Plant and Cell Physiology  2009;50(6):1041-1048.
Actin plays fundamental roles in a wide array of plant functions, including cell division, cytoplasmic streaming, cell morphogenesis and organelle motility. Imaging the actin cytoskeleton in living cells is a powerful methodology for studying these important phenomena. Several useful probes for live imaging of filamentous actin (F-actin) have been developed, but new versatile probes are still needed. Here, we report the application of a new probe called Lifeact for visualizing F-actin in plant cells. Lifeact is a short peptide comprising 17 amino acids that was derived from yeast Abp140p. We used a Lifeact–Venus fusion protein for staining F-actin in Arabidopsis thaliana and were able to observe dynamic rearrangements of the actin meshwork in root hair cells. We also used Lifeact–Venus to visualize the actin cytoskeleton in the liverwort Marchantia polymorpha; this revealed unique and dynamic F-actin motility in liverwort cells. Our results suggest that Lifeact could be a useful tool for studying the actin cytoskeleton in a wide range of plant lineages.
PMCID: PMC2694730  PMID: 19369273
Actin; Arabidopsis thaliana; Lifeact; Liverwort; Marchantia polymorpha
21.  The Assembly Pathway of the 19S Regulatory Particle of the Yeast 26S Proteasome 
Molecular Biology of the Cell  2007;18(2):569-580.
The 26S proteasome consists of the 20S proteasome (core particle) and the 19S regulatory particle made of the base and lid substructures, and it is mainly localized in the nucleus in yeast. To examine how and where this huge enzyme complex is assembled, we performed biochemical and microscopic characterization of proteasomes produced in two lid mutants, rpn5-1 and rpn7-3, and a base mutant ΔN rpn2, of the yeast Saccharomyces cerevisiae. We found that, although lid formation was abolished in rpn5-1 mutant cells at the restrictive temperature, an apparently intact base was produced and localized in the nucleus. In contrast, in ΔN rpn2 cells, a free lid was formed and localized in the nucleus even at the restrictive temperature. These results indicate that the modules of the 26S proteasome, namely, the core particle, base, and lid, can be formed and imported into the nucleus independently of each other. Based on these observations, we propose a model for the assembly process of the yeast 26S proteasome.
PMCID: PMC1783769  PMID: 17135287
22.  Blockade of high mobility group box-1 protein attenuates experimental severe acute pancreatitis 
AIM: To examine the effects of anti-high mobility group box 1 (HMGB1) neutralizing antibody in experimental severe acute pancreatitis (SAP).
METHODS: SAP was induced by creating closed duodenal loop in C3H/HeN mice. SAP was induced immediately after intraperitoneal injection of anti-HMGB1 neutralizing antibody (200 μg). Severity of pancreatitis, organ injury (liver, kidney and lung), and bacterial translocation to pancreas was examined 12 h after induction of SAP.
RESULTS: Anti-HMGB1 neutralizing antibody significantly improved the elevation of the serum amylase level and the histological alterations of pancreas and lung in SAP. Anti-HMGB1 antibody also significantly ameliorated the elevations of serum alanine aminotransferase and creatinine in SAP. However, anti-HMGB1 antibody worsened the bacterial translocation to pancreas.
CONCLUSION: Blockade of HMGB1 attenuated the development of SAP and associated organ dysfunction, suggesting that HMGB1 may act as a key mediator for inflammatory response and organ injury in SAP.
PMCID: PMC4088050  PMID: 17171797
Severe acute pancreatitis; High mobility group box-1; Neutralizing antibody; Inflammatory response; Organ dysfunction; Bacterial translocation
23.  Cdc50p, a Conserved Endosomal Membrane Protein, Controls Polarized Growth in Saccharomyces cerevisiae 
Molecular Biology of the Cell  2003;14(2):730-747.
During the cell cycle of the yeast Saccharomyces cerevisiae, the actin cytoskeleton and the growth of cell surface are polarized, mediating bud emergence, bud growth, and cytokinesis. We identified CDC50 as a multicopy suppressor of the myo3 myo5-360 temperature-sensitive mutant, which is defective in organization of cortical actin patches. The cdc50 null mutant showed cold-sensitive cell cycle arrest with a small bud as reported previously. Cortical actin patches and Myo5p, which are normally localized to polarization sites, were depolarized in the cdc50 mutant. Furthermore, actin cables disappeared, and Bni1p and Gic1p, effectors of the Cdc42p small GTPase, were mislocalized in the cdc50 mutant. As predicted by its amino acid sequence, Cdc50p appears to be a transmembrane protein because it was solubilized from the membranes by detergent treatment. Cdc50p colocalized with Vps21p in endosomal compartments and was also localized to the class E compartment in the vps27 mutant. The cdc50 mutant showed defects in a late stage of endocytosis but not in the internalization step. It showed, however, only modest defects in vacuolar protein sorting. Our results indicate that Cdc50p is a novel endosomal protein that regulates polarized cell growth.
PMCID: PMC150004  PMID: 12589066
24.  Amiloride-blockable acid-sensing ion channels are leading acid sensors expressed in human nociceptors 
The Journal of Clinical Investigation  2002;110(8):1185-1190.
Many painful inflammatory and ischemic conditions such as rheumatoid arthritis, cardiac ischemia, and exhausted skeletal muscles are accompanied by local tissue acidosis. In such acidotic states, extracellular protons provoke the pain by opening cation channels in nociceptors. It is generally believed that a vanilloid receptor subtype-1 (VR1) and an acid-sensing ion channel (ASIC) mediate the greater part of acid-induced nociception in mammals. Here we provide evidence for the involvement of both channels in acid-evoked pain in humans and show their relative contributions to the nociception. In our psychophysical experiments, direct infusion of acidic solutions (pH ≥ 6.0) into human skin caused localized pain, which was blocked by amiloride, an inhibitor of ASICs, but not by capsazepine, an inhibitor of VR1. Under more severe acidification (pH 5.0) amiloride was less effective in reducing acid-evoked pain. In addition, capsazepine had a partial blocking effect under these conditions. Amiloride itself neither blocked capsaicin-evoked localized pain in human skin nor inhibited proton-induced currents in VR1-expressing Xenopus oocytes. Our results suggest that ASICs are leading acid sensors in human nociceptors and that VR1 participates in the nociception mainly under extremely acidic conditions.
PMCID: PMC150796  PMID: 12393854
25.  Multiple Roles of Arf1 GTPase in the Yeast Exocytic and Endocytic Pathways 
Molecular Biology of the Cell  2001;12(1):221-238.
ADP-ribosylation factors, a family of small GTPases, are believed to be key regulators of intracellular membrane traffic. However, many biochemical in vitro experiments have led to different models for their involvement in various steps of vesicular transport, and their precise role in living cells is still unclear. We have taken advantage of the powerful yeast genetic system and screened for temperature-sensitive (ts) mutants of the ARF1 gene from Saccharomyces cerevisiae. By random mutagenesis of the whole open reading frame of ARF1 by error-prone PCR, we isolated eight mutants and examined their phenotypes. arf1 ts mutants showed a variety of transport defects and morphological alterations in an allele-specific manner. Furthermore, intragenic complementation was observed between certain pairs of mutant alleles, both for cell growth and intracellular transport. These results demonstrate that the single Arf1 protein is indeed involved in many different steps of intracellular transport in vivo and that its multiple roles may be dissected by the mutant alleles we constructed.
PMCID: PMC30579  PMID: 11160834

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