Colocalization of fluorescent markers in confocal microscope images of plant cells

French, A. P.; Mills, Steven; Swarup, Ranjan; Bennett, Malcolm J.; Pridmore, Tony

doi:10.1038/nprot.2008.31

Cited by 349 publications

(332 citation statements)

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“…As shown in Figures 5A to 5C, the punctate compartments of GPA3-GFP were obviously distinct from the Golgi but partially overlapped with the TGN and PVC. Furthermore, correlation analysis using the Pearson-Spearman correlation (PSC) plugin for ImageJ (French et al, 2008) revealed strong correlation between GPA3-GFP and the PVC marker (r s = 0.646), but the correlation between GPA3-GFP and the TGN marker appeared to be weaker (r s = 0.194).…”

Section: Gpa3 Protein Is Localized To Post-golgi Compartmentsmentioning

confidence: 99%

GLUTELIN PRECURSOR ACCUMULATION3Encodes a Regulator of Post-Golgi Vesicular Traffic Essential for Vacuolar Protein Sorting in Rice Endosperm

et al. 2014

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In seed plants, a major pathway for sorting of storage proteins to the protein storage vacuole (PSV) depends on the Golgi-derived dense vesicles (DVs). However, the molecular mechanisms regulating the directional trafficking of DVs to PSVs remain largely elusive. Here, we report the functional characterization of the rice (Oryza sativa) glutelin precursor accumulation3 (gpa3) mutant, which exhibits a floury endosperm phenotype and accumulates excess proglutelins in dry seeds. Cytological and immunocytochemistry studies revealed that in the gpa3 mutant, numerous proglutelin-containing DVs are misrouted to the plasma membrane and, via membrane fusion, release their contents into the apoplast to form a new structure named the paramural body. Positional cloning of GPA3 revealed that it encodes a plant-specific kelch-repeat protein that is localized to the trans-Golgi networks, DVs, and PSVs in the developing endosperm. In vitro and in vivo experiments verified that GPA3 directly interacts with the rice Rab5a-guanine exchange factor VPS9a and forms a regulatory complex with Rab5a via VPS9a. Furthermore, our genetic data support the notion that GPA3 acts synergistically with Rab5a and VPS9a to regulate DV-mediated post-Golgi traffic in rice. Our findings provide insights into the molecular mechanisms regulating the plant-specific PSV pathway and expand our knowledge of vesicular trafficking in eukaryotes.

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Section: Gpa3 Protein Is Localized To Post-golgi Compartmentsmentioning

confidence: 99%

GLUTELIN PRECURSOR ACCUMULATION3Encodes a Regulator of Post-Golgi Vesicular Traffic Essential for Vacuolar Protein Sorting in Rice Endosperm

et al. 2014

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“…Image analysis was undertaken using the ImageJ analysis program (Abramoff et al, 2004) using the PSC co-localization plug-in (French et al, 2008) to calculate co-localization. At least 20 cells were analysed for each condition.…”

Section: Location Of Dengue Virus Replication Complexmentioning

confidence: 99%

Co-localization of constituents of the dengue virus translation and replication machinery with amphisomes

Panyasrivanit

Khakpoor

Wikan

et al. 2009

Journal of General Virology

149

176

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Infections with dengue virus (DENV) are a significant public health concern in tropical and subtropical regions. However, little detail is known about how DENV interacts with the host-cell machinery to facilitate its translation and replication. In DENV-infected HepG2 cells, an increase in the level of LC3-II (microtubule-associated protein 1 light chain 3 form II), the autophagosomal membrane-bound form of LC3, was observed, and LC3 was found to co-localize with dsRNA and DENV NS1 protein, as well as ribosomal protein L28, indicating the presence of at least some of the DENV translation/replication machinery on autophagic vacuoles. Inhibition of fusion of autophagic vacuoles with lysosomes resulted in an increase in both intracellular and extracellular virus, and co-localization observed between mannose-6-phosphate receptor (MPR) and dsRNA and between MPR and LC3 identified the autophagic vacuoles as amphisomes. Amphisomes are formed as a result of fusion between endosomal and autophagic vacuoles, and as such provide a direct link between virus entry and subsequent replication and translation. INTRODUCTIONWith an estimated 100 million infections per year worldwide, dengue virus (DENV), which is spread to humans by the bite of female Aedes mosquitoes, represents a significant public health threat in tropical and subtropical countries (Guzman & Kouri, 2002). The DENV complex comprises four antigenically distinct viruses termed DENV serotypes 1, 2, 3 and 4 (DENV-1 to -4), all of which can cause a wide spectrum of disease presentation, from a relatively mild febrile disease to a life-threatening haemorrhagic syndrome (Malavige et al., 2004). DENV is an enveloped, positivesense, single-stranded RNA virus of approximately 11 kb that encodes three structural proteins (core, pre-membrane and envelope) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) in one open reading frame (Chang, 1997). Entry of DENV into a susceptible cell occurs primarily through receptormediated endocytosis via clathrin-coated pits (Krishnan et al., 2007) and the virus is then trafficked to endosomes (Krishnan et al., 2007;van der Schaar et al., 2007) and fuses with the endosomal membranes (Heinz et al., 2004) through a pH-dependent conformational change in the envelope protein (Modis et al., 2004;Mukhopadhyay et al., 2005). The fate of the released nucleocapsid is largely obscure, although it is known that, after uncoating of the virus genome, the host-cell translational machinery is utilized to synthesize a polyprotein precursor that is processed co-and post-translationally by a virus-encoded protease (NS3) and host signallases (Cahour et al., 1992). Translation and subsequent replication of the DENV genome occur in tight association with intracellular membranous structures that are believed to be endoplasmic reticulum (ER)-derived (Clyde et al., 2006;Miller & Krijnse-Locker, 2008;Salonen et al., 2005), although the exact origin of these membranes remains unclear.Autophagy is a lysosomal degradation pathway involve...

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Section: Nuclear Retention Of Il-1a By Immobilizationmentioning

confidence: 99%

“…Pearson's co-localization analysis was used to assess the co-localization of IL-1a-GFP with nuclear proteins and DAPI [20].…”

Section: Confocal Microscopy and Frapmentioning

confidence: 99%

“…Co-localization was quantified using Pearson's co-localization coefficient, which assesses the strength of linear correlation between pixel fluorescence in two different channels. 11 indicates perfect co-localization, a value of zero indicates no co-localization, and À1 indicates a perfect negative correlation between the intensities in the two channels [20].The IL-1a N-terminal domain has been reported previously to localize to SC-35 1 nuclear speckles (storage sites for RNA splicing factors [13,21]]. We found that IL-1a-GFP similarly became partially restricted to SC-35 1 nuclear speckles in ATP-depleted cells (Fig.…”

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Nuclear retention of IL‐1α by necrotic cells: A mechanism to dampen sterile inflammation

2009

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Sterile inflammation is a host response to tissue injury that is mediated by damage-associated molecular patterns released from dead cells. Sterile inflammation worsens damage in a number of injury paradigms. The pro-inflammatory cytokine IL-1a is reported to be a damage-associated molecular pattern released from dead cells, and it is known to exacerbate brain injury caused by stroke. In the brain, IL-1a is produced by microglia, the resident brain macrophages. We found that IL-1a is actively trafficked to the nuclei of microglia, and hence tested the hypothesis that trafficking of IL-1a to the nucleus would inhibit its release following necrotic cell death, limiting sterile inflammation. Microglia subjected to oxygenglucose deprivation died via necrosis. Under these conditions, microglia expressing nuclear IL-1a released significantly less IL-1a than microglia with predominantly cytosolic IL-1a. The remaining IL-1a was immobilized in the nuclei of the dead cells. Thus, nuclear retention of IL-1a may serve to limit inflammation following cell death.Key words: IL-1a . Microglia . Necrosis . Nuclear retention . Sterile inflammation Supporting Information available online IntroductionSterile inflammation is a host response to tissue injury that can exacerbate the initial insult [1]. Intracellular molecules released from necrotic cells act as damage-associated molecular patterns (DAMP), signaling to the innate immune system that tissue injury has occurred [2]. IL-1a, a pro-inflammatory member of the IL-1 cytokine family [3], has recently been identified as a DAMP released from necrotic cells [4]. In addition, necrotic cell DAMP are reported to stimulate IL-1a production by immune cells, which further exacerbates sterile inflammation [1]. Experimental stroke in rodents elicits an inflammatory response that markedly exacerbates brain injury [5]. IL-1a is a key contributor to this injury, and its genetic ablation (along with IL-1b, a related cytokine) causes a 70% reduction in the brain injury caused by stroke [6].In the brain after injury (e.g. stroke, hemorrhage, trauma), IL-1 family cytokines are produced by microglia, the resident brain macrophages [7]. IL-1a is produced in the cytosol as a 31-kDa protein that, following release from necrotic cells, activates the type I IL-1 receptor on responsive cell membranes [8,9]. The Nterminal domain of IL-1a contains a nuclear localization sequence, which mediates active nuclear import [10,11]. IL-1a has been reported to act within the nucleus to regulate cytokine transcription and RNA splicing [12,13]. In this study we sought to test the hypothesis that IL-1a nuclear import also inhibits IL-1a release following necrotic cell death. We report that IL-1a nuclear import and intranuclear retention reduce IL-1a release from necrotic microglia. Thus, IL-1a nuclear retention by necrotic cells may inhibit injury-induced inflammation. Results IL-1a nuclear localization is inhibited at high cell densityTo investigate the effects of IL-1a nuclear localization on IL-1a release after ne...

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Colocalization of fluorescent markers in confocal microscope images of plant cells

Cited by 349 publications

References 20 publications

GLUTELIN PRECURSOR ACCUMULATION3Encodes a Regulator of Post-Golgi Vesicular Traffic Essential for Vacuolar Protein Sorting in Rice Endosperm

GLUTELIN PRECURSOR ACCUMULATION3Encodes a Regulator of Post-Golgi Vesicular Traffic Essential for Vacuolar Protein Sorting in Rice Endosperm

Co-localization of constituents of the dengue virus translation and replication machinery with amphisomes

Nuclear retention of IL‐1α by necrotic cells: A mechanism to dampen sterile inflammation

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