Tandem affinity purification coupled to mass spectrometry (TAP-MS) is one of the most advanced methods to characterize protein complexes in plants, giving a comprehensive view on the protein-protein ...interactions (PPIs) of a certain protein of interest (bait). The bait protein is fused to a double affinity tag, which consists of a protein G tag and a streptavidin-binding peptide separated by a very specific protease cleavage site, allowing highly specific protein complex isolation under near-physiological conditions. Implementation of this optimized TAP tag, combined with ultrasensitive MS, means that these experiments can be performed on small amounts (25 mg of total protein) of protein extracts from Arabidopsis cell suspension cultures. It is also possible to use this approach to isolate low abundant protein complexes from Arabidopsis seedlings, thus opening perspectives for the exploration of protein complexes in a plant developmental context. Next to protocols for efficient biomass generation of seedlings (∼7.5 months), we provide detailed protocols for TAP (1 d), and for sample preparation and liquid chromatography-tandem MS (LC-MS/MS; ∼5 d), either from Arabidopsis seedlings or from cell cultures. For the identification of specific co-purifying proteins, we use an extended protein database and filter against a list of nonspecific proteins on the basis of the occurrence of a co-purified protein among 543 TAP experiments. The value of the provided protocols is illustrated through numerous applications described in recent literature.
Clathrin-mediated endocytosis is the major mechanism for eukaryotic plasma membrane-based proteome turn-over. In plants, clathrin-mediated endocytosis is essential for physiology and development, but ...the identification and organization of the machinery operating this process remains largely obscure. Here, we identified an eight-core-component protein complex, the TPLATE complex, essential for plant growth via its role as major adaptor module for clathrin-mediated endocytosis. This complex consists of evolutionarily unique proteins that associate closely with core endocytic elements. The TPLATE complex is recruited as dynamic foci at the plasma membrane preceding recruitment of adaptor protein complex 2, clathrin, and dynamin-related proteins. Reduced function of different complex components severely impaired internalization of assorted endocytic cargoes, demonstrating its pivotal role in clathrin-mediated endocytosis. Taken together, the TPLATE complex is an early endocytic module representing a unique evolutionary plant adaptation of the canonical eukaryotic pathway for clathrin-mediated endocytosis.
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•A multimeric protein complex is essential for plant clathrin-mediated endocytosis•The TPLATE complex is essential and functions in concert with the AP2 complex•Impaired TPLATE complex function abates internalization of several cargo molecules•The TPLATE complex represents an evolutionarily unique strategy to internalize cargo
The characterization of TPLATE, a plant-specific complex involved in clathrin-mediated endocytosis, highlights an evolutionary adaptation of an essential endocytic pathway in plants.
Plant bZIP group I transcription factors have been reported mainly for their role during vascular development and osmosensory responses. Interestingly, bZIP29 has been identified in a cell cycle ...interactome, indicating additional functions of bZIP29 in plant development. Here, bZIP29 was functionally characterized to study its role during plant development. It is not present in vascular tissue but is specifically expressed in proliferative tissues. Genome-wide mapping of bZIP29 target genes confirmed its role in stress and osmosensory responses, but also identified specific binding to several core cell cycle genes and to genes involved in cell wall organization. bZIP29 protein complex analyses validated interaction with other bZIP group I members and provided insight into regulatory mechanisms acting on bZIP dimers. In agreement with bZIP29 expression in proliferative tissues and with its binding to promoters of cell cycle regulators, dominant-negative repression of bZIP29 altered the cell number in leaves and in the root meristem. A transcriptome analysis on the root meristem, however, indicated that bZIP29 might regulate cell number through control of cell wall organization. Finally, ectopic dominant-negative repression of bZIP29 and redundant factors led to a seedling-lethal phenotype, pointing to essential roles for bZIP group I factors early in plant development.
The anaphase-promoting complex/cyclosome (APC/C) is a large multiprotein E3 ubiquitin ligase involved in ubiquitin-dependent proteolysis of key cell cycle regulatory proteins, including the ...destruction of mitotic cyclins at the metaphase-to-anaphase transition. Despite its importance, the role of the APC/C in plant cells and the regulation of its activity during cell division remain poorly understood. Here, we describe the identification of a plant-specific negative regulator of the APC/C complex, designated SAMBA. In Arabidopsis thaliana , SAMBA is expressed during embryogenesis and early plant development and plays a key role in organ size control. Samba mutants produced larger seeds, leaves, and roots, which resulted from enlarged root and shoot apical meristems, and, additionally, they had a reduced fertility attributable to a hampered male gametogenesis. Inactivation of SAMBA stabilized A2-type cyclins during early development. Our data suggest that SAMBA regulates cell proliferation during early development by targeting CYCLIN A2 for APC/C-mediated proteolysis.
Proteins are the cell’s functional entities. Rather than operating independently, they interact with other proteins. Capturing in vivo protein complexes is therefore crucial to gain understanding of ...the function of a protein in a cellular context. Affinity purification coupled to mass spectrometry has proven to yield a wealth of information about protein complex constitutions for a broad range of organisms. For
Oryza sativa
, the technique has been initiated in callus and shoots, but has not been optimized ever since. We translated an optimized tandem affinity purification (TAP) approach from
Arabidopsis thaliana
toward
Oryza sativa
, and demonstrate its applicability in a variety of rice tissues. A list of non-specific and false positive interactors is presented, based on re-occurrence over more than 170 independent experiments, to filter bona fide interactors. We demonstrate the sensitivity of our approach by isolating the complexes for the rice ANAPHASE PROMOTING COMPLEX SUBUNIT 10 (APC10) and CYCLIN-DEPENDENT KINASE D (CDKD) proteins from the proliferation zone of the emerging fourth leaf. Next to APC10 and CDKD, we tested several additional baits in the different rice tissues and reproducibly retrieved at least one interactor for 81.4 % of the baits screened for in callus tissue and T1 seedlings. By transferring an optimized TAP tag combined with state-of-the-art mass spectrometry, our TAP protocol enables the discovery of interactors for low abundance proteins in rice and opens the possibility to capture complex dynamics by comparing tissues at different stages of a developing rice organ.
Physical education (PE) teachers have a physically demanding job, putting them at a considerable risk for musculoskeletal injuries. To structurally develop tailored injury prevention programmes for ...PE teachers, a clear understanding of the extent, characteristics and underlying factors of their musculoskeletal injuries compared to referents is necessary. Therefore, the current study prospectively followed 103 PE teachers and 58 non-PE teachers, who registered musculoskeletal injuries and time of exposure to sports participation during one school year. Pearson x^2-tests and independent samples t-tests determined significant differences between PE and non-PE teachers regarding demographics and variables possibly related to injury occurrence. PE teachers had 1.23 and non-PE teachers 0.78 injuries/teacher/school year. This difference was significantly different after adjustment for hours spent weekly on intracurricular teaching during the career and for injury history during the preceding six months (P = 0.009; OR = 0.511; 95% CI = 0.308-0.846). PE teachers' most affected body parts were the knee and the back. PE teachers had a more extensive injury history (P ( 0.001), a higher work- (P ( 0.001) and sport index (P ( 0.001), practiced more sports (P ( 0.002) and taught more extracurricular sports (P = 0.001). Future injury prevention programmes should take account for the great injury history and heavy physical load in PE teachers. (Autor).
Physical education (PE) teachers are at a high risk of musculoskeletal sports or work-related injuries because of the physical activity as inherent part of their profession. Such injuries have a ...negative impact on work and leisure time activities, and effective injury prevention interventions are needed. The present study aimed at testing the effectiveness of an injury prevention intervention that was developed and optimized according to PE teachers' wishes and values. Fifty-five PE teachers were randomly assigned to intervention or control group. Intervention group teachers engaged in two days of training during which they familiarized with eight injury prevention strategies (seven intrinsic and one extrinsic). A special feature of the intervention was that the way of delivery was based on the self-determination theory in order to stimulate participants' motivation to adhere to the proposed strategies. Prospective registrations during one school year were conducted concerning injuries and preventive behaviours. Results showed that the intervention group teachers had a lower number of injuries per 1000 h time of exposure (TOE) than the controls (INT: 0.49, CON: 1.14 injuries/1000 h TOE, OR: 2.32, 95% CI: 1.06-5.07), and applied a broader variety of strategies including dynamic and static stretching, core stability, balance and strength training, when compared to the controls who mainly engaged in warming-up. In conclusion, with the same amount of time, an injury reduction was found in PE teachers through a more balanced use of provided preventive strategies.
Background: Injury prevention is highly needed in physically active populations, such as pre-service and in-service physical education teachers (PETs). As a lack of adherence to preventive strategies ...is problematic in injury, it seems crucial to develop and optimize interventions that correspond to the specific needs and wishes of PETs.
Aim: The purpose of the present study was fourfold. Specifically, we aimed at (1) systematically optimizing an injury prevention intervention for PETs, based on teachers' qualitative evaluation of the intervention, (2) quantitatively investigating whether the appreciation of the injury prevention intervention was higher after optimization, (3) examining whether participation in the intervention resulted in positive changes in teachers' perceived utility of, and confidence to apply the proposed preventive strategies, as well as their knowledge about these strategies, and (4) describing teachers' adherence to the proposed preventive strategies while they were engaging in the intervention.
Participants: Twenty PETs (13 men, Mage = 42.1 ± 12.17 years) from nine different secondary schools in Flanders (Belgium) voluntarily participated in this study. The intervention, based on findings from the continuing professional development (CPD) literature, and the principles of the self-determination theory, took place on two different training days (Training A and Training B) and consisted of seven intrinsic injury prevention strategies.
Data collection and analysis: Qualitative data on teachers' appreciation of the intervention were collected by means of focus group interviews, which were used to optimize the intervention (Aim 1). Quantitative data on teachers' appreciation were collected directly after they had engaged in the intervention and were used to compare the appreciation from the initial to more optimized versions of the trainings (Aim 2). Further, by means of repeated measures ANOVAs positive evolutions in teachers' belief in the utility of, confidence to apply and knowledge about the provided strategies across time were examined (Aim 3). Finally, teachers' adherence was evaluated by relying on weekly online registrations of minutes and types of preventive strategies teachers had practised during the past week (Aim 4).
Findings: Despite the relatively high initial appreciation scores (4.2 out of 5), the optimized versions of the trainings scored better on interaction, intelligibility, innovation, practical usefulness, and recommendation (Aims 1 and 2). Significant differences were found over time in terms of within-teacher changes in perceived utility of (4.05-4.73-4.48; p < .001), confidence to apply (3.75-3.96-4.26; p < .001) and knowledge about preventive strategies (2.49-3.53-3.39; p < .001; Aim 3). Finally, prospective registrations of teachers' adherence to the proposed strategies showed a mean time of 62.1 ± 48.6 min/week with a balanced distribution of the different strategies (Aim 4).
Conclusions: The present study developed an intervention that optimally fulfils the wishes and needs of the target population, namely PETs, and can readily be implemented in PET education programmes or CPD programmes for in-service PETs. Results of this pilot study are promising because teachers displayed increases in several relevant outcomes. Various suggestions are formulated on how to increase PETs' appreciation of CPD programmes.
De eurocrisis in het nieuws Joris, Willem; d'Haenens, Leen; Van Gorp, Baldwin ...
Tijdschrift voor communicatiewetenschap,
06/2013, Letnik:
41, Številka:
2
Journal Article
Recenzirano
European debt crisis in the news. A frame analysis of Flemish newspaper coverage This research examines the news frames used in the news coverage about the European debt crisis. The mainstream media ...are the main source of information for the European citizens concerning this crisis in the Eurozone. Therefore, it is interesting to research the news coverage through an in-depth content analysis. First, we conducted an inductive analysis to recognize the dominant news frames about this issue. Three dominant frames were identified: conflict, disease and natural disaster. Secondly, we executed a deductive analysis to measure the frequency of these frames. The conflict frame appeared the most, followed by the disease frame and the natural disaster frame. In this article we studied the news coverage in Flemish newspapers. However, this research is the first part of a larger study that will analyze more EU member states.
De eurocrisis in het nieuws Joris, Willem; d’Haenens, Leen; Van Gorp, Baldwin ...
Tijdschrift voor communicatiewetenschap,
06/2013, Letnik:
41, Številka:
2
Journal Article
Recenzirano
European debt crisis in the news. A frame analysis of Flemish newspaper coverage
European debt crisis in the news. A frame analysis of Flemish newspaper coverage
This research examines the news ...frames used in the news coverage about the European debt crisis. The mainstream media are the main source of information for the European citizens concerning this crisis in the Eurozone. Therefore, it is interesting to research the news coverage through an in-depth content analysis. First, we conducted an inductive analysis to recognize the dominant news frames about this issue. Three dominant frames were identified: conflict, disease and natural disaster. Secondly, we executed a deductive analysis to measure the frequency of these frames. The conflict frame appeared the most, followed by the disease frame and the natural disaster frame. In this article we studied the news coverage in Flemish newspapers. However, this research is the first part of a larger study that will analyze more EU member states.
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