scholarly journals The Anaphase Promoting Complex/Cyclosome Subunit 11 and Its Role in Organ Size and Plant Development

2021 ◽  
Vol 12 ◽  
Author(s):  
Rodrigo Porto Schwedersky ◽  
Marina de Lyra Soriano Saleme ◽  
Ingrid Andrade Rocha ◽  
Patricia da Fonseca Montessoro ◽  
Adriana Silva Hemerly ◽  
...  
Keyword(s):  
Plant Development ◽  
Fluorescent Protein ◽  
Cortical Cell ◽  
Quiescent Center ◽  
Anaphase Promoting Complex ◽  
Catalytic Subunits ◽  
Fluorescent Marker ◽  
Green Fluorescent ◽  
Mutant Lines

The anaphase promoting complex/cyclosome (APC/C), a member of the E3 ubiquitin ligase family, plays an important role in recognizing the substrates to be ubiquitylated. Progression of anaphase, and therefore, of the cell cycle, is coordinated through cyclin degradation cycles dependent on proteolysis triggered by APC/C. The APC/C activity depends on the formation of a pocket comprising the catalytic subunits, APC2, APC11, and APC10. Among these, the role of APC11 outside the cell division cycle is poorly understood. Therefore, the goal of this work was to analyze the function of APC11 during plant development by characterizing apc11 knock-down mutant lines. Accordingly, we observed decreased apc11 expression in the mutant lines, followed by a reduction in meristem root size based on the cortical cell length, and an overall size diminishment throughout the development. Additionally, crosses of apc11-1 and amiR-apc11 with plants carrying a WUSCHEL-RELATED HOMEOBOX5 (WOX5) fluorescent marker showed a weakening of the green fluorescent protein-positive cells in the Quiescent Center. Moreover, plants with apc11-1 show a decreased leaf area, together with a decrease in the cell area when the shoot development was observed by kinematics analysis. Finally, we observed a decreased APC/C activity in the root and shoot meristems in crosses of pCYCB1;1:D-box-GUS with apc11-1 plants. Our results indicate that APC11 is important in the early stages of development, mediating meristematic architecture through APC/C activity affecting the overall plant growth.

scholarly journals The Role of Callose Deposition Along Plasmodesmata in Nematode Feeding Sites

2010 ◽  
Vol 23 (5) ◽  
pp. 549-557 ◽  
Author(s):  
Julia Hofmann ◽  
Mohamed Youssef-Banora ◽  
Janice de Almeida-Engler ◽  
Florian M. W. Grundler
Keyword(s):  
Fluorescent Protein ◽  
Giant Cells ◽  
Cyst Nematode ◽  
Cyst Nematodes ◽  
Callose Deposition ◽  
Movement Proteins ◽  
Green Fluorescent ◽  
Mutant Lines ◽  
Nematode Development

Infective second-stage juveniles of the obligate plant-parasitic root-knot and cyst nematodes invade plant roots to induce specialized feeding structures. Here, we present data on the distribution of plasmodesmata in cell walls of syncytia and giant cells induced by cyst and root-knot nematodes. An Arabidopsis and a tobacco line were used, containing viral movement proteins fused to green fluorescent protein as a localization marker for plasmodesmata. Plasmodesmata were detected in walls between giant cells but also in walls toward neighboring cells. In syncytia, plasmodesmata were mainly detected at later stages. In young syncytia, few plasmodesmata were observed and a specific temporal callose deposition along plasmodesmata indicated impaired symplasmic exchange. In order to study the relevance of callose deposition for successful cyst nematode development in Arabidopsis, two mutant lines inhibited in callose synthesis and degradation, respectively, were used in nematode infection assays. Histological analyses showed that syncytia were smaller when callose degradation was reduced, indicating a significant importance of this process to cyst nematode development.

scholarly journals 20-HETE-mediated cytotoxicity and apoptosis in ischemic kidney epithelial cells

2008 ◽  
Vol 294 (3) ◽  
pp. F562-F570 ◽  
Author(s):  
Vani Nilakantan ◽  
Cheryl Maenpaa ◽  
Guangfu Jia ◽  
Richard J. Roman ◽  
Frank Park
Keyword(s):  
Caspase 3 ◽  
Fluorescent Protein ◽  
Ischemia Reperfusion ◽  
Atp Depletion ◽  
Cellular Damage ◽  
Apoptotic Pathway ◽  
Injury Model ◽  
Green Fluorescent

20-HETE, a metabolite of arachidonic acid, has been implicated as a mediator of free radical formation and tissue death following ischemia-reperfusion (IR) injury in the brain and heart. The present study examined the role of this pathway in a simulated IR renal injury model in vitro. Modified self-inactivating lentiviral vectors were generated to stably overexpress murine Cyp4a12 following transduction into LLC-PK1 cells (LLC-Cyp4a12). We compared the survival of control and transduced LLC-PK1 cells following 4 h of ATP depletion and 2 h of recovery in serum-free medium. ATP depletion-recovery of LLC-Cyp4a12 cells resulted in a significantly higher LDH release ( P < 0.05) compared with LLC-enhanced green fluorescent protein (EGFP) cells. Treatment with the SOD mimetic MnTMPyP (100 μM) resulted in decreased cytotoxicity in LLC-Cyp4a12 cells. The selective 20-HETE inhibitor HET-0016 (10 μM) also inhibited cytotoxicity significantly ( P < 0.05) in LLC-Cyp4a12 cells. Dihydroethidium fluorescence showed that superoxide levels were increased to the same degree in LLC-EGFP and LLC-Cyp4a12 cells after ATP depletion-recovery compared with control cells and that this increase was inhibited by MnTMPyP. There was a significant increase ( P < 0.05) of caspase-3 cleavage, an effector protease of the apoptotic pathway, in the LLC-Cyp4a12 vs. LLC-EGFP cells ( P < 0.05). This was abolished in the presence of HET-0016 ( P < 0.05) or MnTMPyP ( P < 0.01). These results demonstrate that 20-HETE overexpression can significantly exacerbate the cellular damage that is associated with renal IR injury and that the programmed cell death is mediated by activation of caspase-3 and is partially dependent on enhanced CYP4A generation of free radicals.

Defining the Role of Arginine 96 in Green Fluorescent Protein Fluorophore Biosynthesis†,‡

Biochemistry ◽  
2005 ◽  
Vol 44 (49) ◽  
pp. 16211-16220 ◽  
Author(s):  
Timothy I. Wood ◽  
David P. Barondeau ◽  
Chiharu Hitomi ◽  
Carey J. Kassmann ◽  
John A. Tainer ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent

scholarly journals Targeting of Protein Kinase C-ϵ during Fcγ Receptor-dependent Phagocytosis Requires the ϵC1B Domain and Phospholipase C-γ1

2006 ◽  
Vol 17 (2) ◽  
pp. 799-813 ◽  
Author(s):  
Keylon L. Cheeseman ◽  
Takehiko Ueyama ◽  
Tanya M. Michaud ◽  
Kaori Kashiwagi ◽  
Demin Wang ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Phospholipase D ◽  
Fluorescent Protein ◽  
Wild Type ◽  
Fcγ Receptor ◽  
Kinase C ◽  
Green Fluorescent

Protein kinase C-ϵ (PKC-ϵ) translocates to phagosomes and promotes uptake of IgG-opsonized targets. To identify the regions responsible for this concentration, green fluorescent protein (GFP)-protein kinase C-ϵ mutants were tracked during phagocytosis and in response to exogenous lipids. Deletion of the diacylglycerol (DAG)-binding ϵC1 and ϵC1B domains, or the ϵC1B point mutant ϵC259G, decreased accumulation at phagosomes and membrane translocation in response to exogenous DAG. Quantitation of GFP revealed that ϵC259G, ϵC1, and ϵC1B accumulation at phagosomes was significantly less than that of intact PKC-ϵ. Also, the DAG antagonist 1-hexadecyl-2-acetyl glycerol (EI-150) blocked PKC-ϵ translocation. Thus, DAG binding to ϵC1B is necessary for PKC-ϵ translocation. The role of phospholipase D (PLD), phosphatidylinositol-specific phospholipase C (PI-PLC)-γ1, and PI-PLC-γ2 in PKC-ϵ accumulation was assessed. Although GFP-PLD2 localized to phagosomes and enhanced phagocytosis, PLD inhibition did not alter target ingestion or PKC-ϵ localization. In contrast, the PI-PLC inhibitor U73122 decreased both phagocytosis and PKC-ϵ accumulation. Although expression of PI-PLC-γ2 is higher than that of PI-PLC-γ1, PI-PLC-γ1 but not PI-PLC-γ2 consistently concentrated at phagosomes. Macrophages from PI-PLC-γ2-/-mice were similar to wild-type macrophages in their rate and extent of phagocytosis, their accumulation of PKC-ϵ at the phagosome, and their sensitivity to U73122. This implicates PI-PLC-γ1 as the enzyme that supports PKC-ϵ localization and phagocytosis. That PI-PLC-γ1 was transiently tyrosine phosphorylated in nascent phagosomes is consistent with this conclusion. Together, these results support a model in which PI-PLC-γ1 provides DAG that binds to ϵC1B, facilitating PKC-ϵ localization to phagosomes for efficient IgG-mediated phagocytosis.

scholarly journals Analysis of the Bacillus subtilis spoIIIJ Gene and Its Paralogue Gene, yqjG

2002 ◽  
Vol 184 (7) ◽  
pp. 1998-2004 ◽  
Author(s):  
Takako Murakami ◽  
Koki Haga ◽  
Michio Takeuchi ◽  
Tsutomu Sato
Keyword(s):  
Bacillus Subtilis ◽  
Membrane Proteins ◽  
Vegetative Growth ◽  
Fluorescent Protein ◽  
Specific Expression ◽  
Rich Media ◽  
Green Fluorescent ◽  
High Level ◽  
Fluorescent Protein Reporter

ABSTRACT The Bacillus subtilis spoIIIJ gene, which has been proven to be vegetatively expressed, has also been implicated as a sporulation gene. Recent genome sequencing information in many organisms reveals that spoIIIJ and its paralogous gene, yqjG, are conserved from prokaryotes to humans. A homologue of SpoIIIJ/YqjG, the Escherichia coli YidC is involved in the insertion of membrane proteins into the lipid bilayer. On the basis of this similarity, it was proposed that the two homologues act as translocase for the membrane proteins. We studied the requirements for spoIIIJ and yqjG during vegetative growth and sporulation. In rich media, the growth of spoIIIJ and yqjG single mutants were the same as that of the wild type, whereas spoIIIJ yqjG double inactivation was lethal, indicating that together these B. subtilis translocase homologues play an important role in maintaining the viability of the cell. This result also suggests that SpoIIIJ and YqjG probably control significantly overlapping functions during vegetative growth. spoIIIJ mutations have already been established to block sporulation at stage III. In contrast, disruption of yqjG did not interfere with sporulation. We further show that high level expression of spoIIIJ during vegetative phase is dispensable for spore formation, but the sporulation-specific expression of spoIIIJ is necessary for efficient sporulation even at the basal level. Using green fluorescent protein reporter to monitor SpoIIIJ and YqjG localization, we found that the proteins localize at the cell membrane in vegetative cells and at the polar and engulfment septa in sporulating cells. This localization of SpoIIIJ at the sporulation-specific septa may be important for the role of spoIIIJ during sporulation.

scholarly journals Inactivation of a Heterocyst-Specific Invertase Indicates a Principal Role of Sucrose Catabolism in Heterocysts of Anabaena sp.

2010 ◽  
Vol 192 (20) ◽  
pp. 5526-5533 ◽  
Author(s):  
Rocío López-Igual ◽  
Enrique Flores ◽  
Antonia Herrero
Keyword(s):  
Fluorescent Protein ◽  
Northern Analysis ◽  
Filamentous Cyanobacterium ◽  
Nitrogen Deprivation ◽  
Vegetative Cells ◽  
Anabaena Sp ◽  
Green Fluorescent ◽  
Pcc 7120 ◽  
Diazotrophic Growth

ABSTRACT Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that carries out N2 fixation in specialized cells called heterocysts, which exchange nutrients and regulators with the filament's vegetative cells that perform the photosynthetic fixation of CO2. The Anabaena genome carries two genes coding for alkaline/neutral invertases, invA and invB. As shown by Northern analysis, both genes were expressed monocistronically and induced under nitrogen deprivation, although induction was stronger for invB than for invA. Whereas expression of an InvA-N-GFP fusion (green fluorescent protein [GFP] fused to the N terminus of the InvA protein [InvA-N]) was homogeneous along the cyanobacterial filament, consistent with the lack of dependence on HetR, expression of an InvB-N-GFP fusion upon combined nitrogen deprivation took place mainly in differentiating and mature heterocysts. In an hetR genetic background, the InvB-N-GFP fusion was strongly expressed all along the filament. An insertional mutant of invA could grow diazotrophically but was impaired in nifHDK induction and exhibited an increased frequency of heterocysts, suggesting a regulatory role of the invertase-mediated carbon flux in vegetative cells. In contrast, an invB mutant was strongly impaired in diazotrophic growth, showing a crucial role of sucrose catabolism mediated by the InvB invertase in the heterocysts.

scholarly journals Mouse Emi2 is required to enter meiosis II by reestablishing cyclin B1 during interkinesis

2006 ◽  
Vol 174 (6) ◽  
pp. 791-801 ◽  
Author(s):  
Suzanne Madgwick ◽  
David V. Hansen ◽  
Mark Levasseur ◽  
Peter K. Jackson ◽  
Keith T. Jones
Keyword(s):  
Fluorescent Protein ◽  
Polar Body ◽  
Cyclin B1 ◽  
Anaphase Promoting Complex ◽  
Morpholino Knockdown ◽  
Short Time ◽  
Mitotic Inhibitor ◽  
Antisense Morpholino ◽  
Meiosis I

During interkinesis, a metaphase II (MetII) spindle is built immediately after the completion of meiosis I. Oocytes then remain MetII arrested until fertilization. In mouse, we find that early mitotic inhibitor 2 (Emi2), which is an anaphase-promoting complex inhibitor, is involved in both the establishment and the maintenance of MetII arrest. In MetII oocytes, Emi2 needs to be degraded for oocytes to exit meiosis, and such degradation, as visualized by fluorescent protein tagging, occurred tens of minutes ahead of cyclin B1. Emi2 antisense morpholino knockdown during oocyte maturation did not affect polar body (PB) extrusion. However, in interkinesis the central spindle microtubules from meiosis I persisted for a short time, and a MetII spindle failed to assemble. The chromatin in the oocyte quickly decondensed and a nucleus formed. All of these effects were caused by the essential role of Emi2 in stabilizing cyclin B1 after the first PB extrusion because in Emi2 knockdown oocytes a MetII spindle was recovered by Emi2 rescue or by expression of nondegradable cyclin B1 after meiosis I.

Functional comparison of the role of dynamin 2 splice variants on GLUT-4 endocytosis in 3T3L1 adipocytes

2000 ◽  
Vol 278 (5) ◽  
pp. E825-E831 ◽  
Author(s):  
Aimee W. Kao ◽  
Chunmei Yang ◽  
Jeffrey E. Pessin
Keyword(s):  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Splice Variants ◽  
Wild Type ◽  
Cell Cytoplasm ◽  
Functional Specificity ◽  
Glut 4 ◽  
Green Fluorescent ◽  
Dynamin 2

Previously, we reported that expression of a dominant-interfering neuronal-specific dynamin 1 (K44A/dynamin 1) inhibited the plasma membrane internalization of GLUT-4 in 3T3L1 adipocytes (15). To investigate the role of the ubiquitously expressed isoform of dynamin, dynamin 2, on adipocyte GLUT-4 internalization, and to determine whether dynamin splice variants have functional specificity, we expressed each of the four dynamin 2 isoforms (aa, ab, ba, and bb) as either wild-type proteins or GTPase-defective mutants. When expressed as enhanced green fluorescent protein (EGFP) fusions, these isoforms were found to have overlapping subcellular distributions being localized throughout the cell cytoplasm, on punctate vesicles and in a perinuclear compartment. This distribution was qualitatively similar to that of endogenous dynamin 2 and overlapped with GLUT-4 in the basal state. Expression of wild-type dynamin 2 isoforms had no effect on the basal or insulin-stimulated distribution of GLUT-4; however, expression of the dominant-interfering dynamin 2 mutants inhibited GLUT-4 endocytosis. These data demonstrate that dynamin 2 is required for GLUT-4 endocytosis in 3T3L1 adipocytes and suggest that, relative to GLUT-4 trafficking, the dynamin 2 splice variants have overlapping functions and are probably not responsible for mediating distinct GLUT-4 budding events.

Sign in / Sign up

Export Citation Format

Share Document