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1.
Eukaryotic cells are characterised by the organised distribution of membrane bounded compartments in their cytoplasm. The endoplasmic reticulum (ER) and the Golgi apparatus (GA) are part of this endomembrane machinery. They are involved in protein flow, and are in charge of specific functions such as the assembly, sorting and transport of newly synthesised proteins, glycoproteins or polysaccharides to their final destination, where the macromolecules are recognised either for action, storage, deposition or degradation. The structural and functional relationship between the ER and GA in higher plants is still a matter of debate. Therefore, it was essential to develop probes that would specifically label proteins or glycoproteins of the endomembrane system in situ. Here we compare two complementary approaches to probe plant endomembranes; immunocytochemistry on fixed cells, and in vivo studies using the expression of GFP tagged chimeric proteins. The structural relationship between ER and GA as based on pharmacological approaches using the two systems is explored. 相似文献
2.
Use of Green Fluorescent Protein (GFP) as a marker has revolutionized biological research in the last few decades. In this brief commentary, we reflect upon the success story of GFP and highlight a few lesser-known facets about GFP that add up to its usefulness. 相似文献
3.
Proteins of the GFP (green fluorescent protein) family have revolutionized life sciences because they allow the tagging of biological samples in a non-invasive genetically encoded way. 'Phototransformable' fluorescent proteins, in particular, have recently attracted widespread interest, as their fluorescence state can be finely tuned by actinic light, a property central to the development of super-resolution microscopy. Beyond microscopy applications, phototransformable fluorescent proteins are also exquisite tools to investigate fundamental protein dynamics. Using light to trigger processes such as photoactivation, photoconversion, photoswitching, blinking and photobleaching allows the exploration of the conformational landscape in multiple directions. In the present paper, we review how structural dynamics of phototransformable fluorescent proteins can be monitored by combining X-ray crystallography, in crystallo optical spectroscopy and simulation tools such as quantum chemistry/molecular mechanics hybrid approaches. Besides their usefulness to rationally engineer better performing fluorescent proteins for nanoscopy and other biotechnological applications, these investigations provide fundamental insights into protein dynamics. 相似文献
4.
Goodin M Yelton S Ghosh D Mathews S Lesnaw J 《Molecular plant-microbe interactions : MPMI》2005,18(7):703-709
Potato yellow dwarf virus (PYDV) and Sonchus yellow net virus (SYNV) belong to the genus Nucleorhabdovirus. These viruses replicate in nuclei of infected cells and mature virions accumulate in the perinuclear space after budding through the inner nuclear membrane. Infection of transgenic Nicotiana benthamiana 16c plants (which constitutively express green fluorescent protein (GFP) targeted to endomembranes) with PYDV or SYNV resulted in virus-specific patterns of accumulation of both GFP and membranes within nuclei. Using immunolocalization and a lipophilic fluorescent dye, we show that the sites of the relocalized membranes were coincident with foci of accumulation of the SYNV nucleocapsid protein. In contrast to the effects of PYDV and SYNV, inoculation of 16c plants with plus-strand RNA viruses did not result in accumulation of intranuclear GFP. Instead, such infections resulted in accumulation of GFP around nuclei, in a manner consistent with proliferation of the endoplasmic reticulum. We propose that the relocalization of GFP in 16c plants can be used to study sites of rhabdovirus accumulation in live cells. This study is the first to use live-cell imaging to characterize the effects of rhabdoviruses on plant nuclear membranes. 相似文献
5.
Falk M 《Trends in cell biology》2002,12(9):399-404
The availability of green fluorescent protein (GFP) as a tracer for observing proteins in living cells has revolutionized cell biology and spurred an intensive search for GFP variants with novel characteristics, additional autofluorescent proteins and alternative techniques of protein labelling. Two recent studies - one on tagging with tetracysteine motifs and labelling with biarsenic fluorophores of different colours, and the other on GFP tagging and fluorescence recovery after photobleaching (FRAP) - show how membrane channels are added and removed from gap junctions by using different fluorescent tags to distinguish between newly synthesized and older protein populations. 相似文献
6.
The secretory membrane system studied in real-time 总被引:2,自引:2,他引:0
Jennifer Lippincott-Schwartz 《Histochemistry and cell biology》2001,116(2):97-107
The discovery and development of green fluorescent protein (GFP) from the jellyfish, Aequorea victoria, has revolutionized studies on protein localization and dynamics by allowing direct observation of a protein's life history and pathway in living cells, previously only deduced from genetic, biochemical, or immunolabeling studies. Applied to the secretory membrane system, which regulates delivery of newly synthesized proteins and lipids to the cell surface, GFP-based studies are providing important new insights into the maintenance and biogenesis of organelles, as well as the origin, pathway, and fate of secretory transport intermediates. 相似文献
7.
Hook SE 《Journal of fish biology》2010,77(9):1999-2022
The advent of any new technology is typically met with great excitement. So it was a few years ago, when the combination of advances in sequencing technology and the development of microarray technology made measurements of global gene expression in ecologically relevant species possible. Many of the review papers published around that time promised that these new technologies would revolutionize environmental biology as they had revolutionized medicine and related fields. A few years have passed since these technological advancements have been made, and the use of microarray studies in non‐model fish species has been adopted in many laboratories internationally. Has the relatively widespread adoption of this technology really revolutionized the fields of environmental biology, including ecotoxicology, aquaculture and ecology, as promised? Or have these studies merely become a novelty and a potential distraction for scientists addressing environmentally relevant questions? In this review, the promises made in early review papers, in particular about the advances that the use of microarrays would enable, are summarized; these claims are compared to the results of recent studies to determine whether the forecasted changes have materialized. Some applications, as discussed in the paper, have been realized and have led to advances in their field, others are still under development. 相似文献
8.
Sebastian Schornack Rene Fuchs Edgar Huitema Ulrich Rothbauer Volker Lipka Sophien Kamoun 《The Plant journal : for cell and molecular biology》2009,60(4):744-754
A key challenge in cell biology is to directly link protein localization to function. The green fluorescent protein (GFP)‐binding protein, GBP, is a 13‐kDa soluble protein derived from a llama heavy chain antibody that binds with high affinity to GFP as well as to some GFP variants such as yellow fluorescent protein (YFP). A GBP fusion to the red fluorescent protein (RFP), a molecule termed a chromobody, was previously used to trace in vivo the localization of various animal antigens. In this study, we extend the use of chromobody technology to plant cells and develop several applications for the in vivo study of GFP‐tagged plant proteins. We took advantage of Agrobacterium tumefaciens‐mediated transient expression assays (agroinfiltration) and virus expression vectors (agroinfection) to express functional GBP:RFP fusion (chromobody) in the model plant Nicotiana benthamiana. We showed that the chromobody is effective in binding GFP‐ and YFP‐tagged proteins in planta. Most interestingly, GBP:RFP can be applied to interfere with the function of GFP fusion protein and to mislocalize (trap) GFP fusions to the plant cytoplasm in order to alter the phenotype mediated by the targeted proteins. Chromobody technology, therefore, represents a new alternative technique for protein interference that can directly link localization of plant proteins to in vivo function. 相似文献
9.
Oomen RJ Séveno-Carpentier E Ricodeau N Bournaud C Conéjéro G Paris N Berthomieu P Marquès L 《The New phytologist》2011,192(1):140-150
? Apart from their antifungal role, plant defensins have recently been shown to be involved in abiotic stress tolerance or in inhibition of root growth when added in plant culture medium. We studied the subcellular localization of these proteins, which may account for these different roles. ? Stable and transient expression of AhPDF1.1::GFP (green fluorescent protein) fusion proteins were analysed in yeast and plants. Functional tests established that the GFP tag did not alter the action of the defensin. Subcellular localization of AhPDF1.1 was characterized: by imaging AhPDF1.1::GFP together with organelle markers; and by immunolabelling AhPDF1.1 in Arabidopsis halleri and Arabidopsis thaliana leaves using a polyclonal serum. ? All our independent approaches demonstrated that AhPDF1.1 is retained in intracellular compartments on the way to the lytic vacuole, instead of being addressed to the apoplasm. ? These findings challenge the commonly accepted idea of secretion of defensins. The subcellular localization highlighted in this study could partly explain the dual role of plant defensins on plant cells and is of major importance to unravel the mechanisms of action of these proteins at the cellular level. 相似文献
10.
G Kotlizky A Katz J van der Laak V Boyko M Lapidot R N Beachy M Heinlein B L Epel 《Molecular plant-microbe interactions : MPMI》2001,14(7):895-904
The Tobacco mosaic virus (TMV) movement protein (MPTMV) mediates cell-to-cell viral trafficking by altering properties of the plasmodesmata (Pd) in infected cells. During the infection cycle, MPTMV becomes transiently associated with endomembranes, microfilaments, and microtubules (MT). It has been shown that the cell-to-cell spread of TMV is reduced in plants expressing the dysfunctional MP mutant MPNT-1. To expand our understanding of the MP function, we analyzed events occurring during the intracellular and intercellular targeting of MPTMV and MPNT-1 when expressed as a fusion protein to green fluorescent protein (GFP), either by biolistic bombardment in a viral-free system or from a recombinant virus. The accumulation of MPTMV:GFP, when expressed in a viral-free system, is similar to MPTMV:GFP in TMV-infected tissues. Pd localization and cell-to-cell spread are late events, occurring only after accumulation of MP:GFP in aggregate bodies and on MT in the target cell. MPNT-1:GFP localizes to MT but does not target to Pd nor does it move cell to cell. The spread of transiently expressed MPTMV:GFP in leaves of transgenic plants that produce MPNT-1 is reduced, and targeting of the MPTMV:GFP to the cytoskeleton is inhibited. Although MPTMV:GFP targets to the Pd in these plants, it is partially impaired for movement. It has been suggested that MPNT-1 interferes with host-dependent processes that occur during the intracellular targeting program that makes MP movement competent. 相似文献
11.
Bridging the gap between omics and earth system science to better understand how environmental change impacts marine microbes
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Thomas Mock Stuart J. Daines Richard Geider Sinead Collins Metodi Metodiev Andrew J. Millar Vincent Moulton Timothy M. Lenton 《Global Change Biology》2016,22(1):61-75
12.
Transgressive segregation and heterosis are the reasons that plant breeding works. Molecular explanations for both phenomena have been suggested and play a contributing role. However, it is often overlooked by molecular genetic researchers that transgressive segregation and heterosis are most simply explained by dispersion of favorable alleles. Therefore, advances in molecular biology will deliver the most impact on plant breeding when integrated with sources of heritable trait variation – and this will be best achieved within a quantitative genetics framework. An example of the power of quantitative approaches is the implementation of genomic selection, which has recently revolutionized animal breeding. Genomic selection is now being applied to both hybrid and inbred crops and is likely to be the major source of improvement in plant breeding practice over the next decade. Breeders’ ability to efficiently apply genomic selection methodologies is due to recent technology advances in genotyping and sequencing. Furthermore, targeted integration of additional molecular data (such as gene expression, gene copy number and methylation status) into genomic prediction models may increase their performance. In this review, we discuss and contextualize a suite of established quantitative genetics themes relating to hybrid vigour, transgressive segregation and their central relevance to plant breeding, with the aim of informing crop researchers outside of the quantitative genetics discipline of their relevance and importance to crop improvement. Better understanding between molecular and quantitative disciplines will increase the potential for further improvements in plant breeding methodologies and so help underpin future food security. 相似文献
13.
The visualization of green fluorescent protein (GFP) fusions with microtubule or actin filament (F-actin) binding proteins has provided new insights into the function of the cytoskeleton during plant development. For studies on actin, GFP fusions to talin have been the most generally used reporters. Although GFP-Talin has allowed in vivo F-actin imaging in a variety of plant cells, its utility in monitoring F-actin in stably transformed plants is limited particularly in developing roots where interesting actin dependent cell processes are occurring. In this study, we created a variety of GFP fusions to Arabidopsis Fimbrin 1 (AtFim1) to explore their utility for in vivo F-actin imaging in root cells and to better understand the actin binding properties of AtFim1 in living plant cells. Translational fusions of GFP to full-length AtFim1 or to some truncated variants of AtFim1 showed filamentous labeling in transient expression assays. One truncated fimbrin-GFP fusion was capable of labeling distinct filaments in stably transformed Arabidopsis roots. The filaments decorated by this construct were highly dynamic in growing root hairs and elongating root cells and were sensitive to actin disrupting drugs. Therefore, the fimbrin-GFP reporters we describe in this study provide additional tools for studying the actin cytoskeleton during root cell development. Moreover, the localization of AtFim1-GFP offers insights into the regulation of actin organization in developing roots by this class of actin cross-linking proteins. 相似文献
14.
Next generation sequencing (NGS) has revolutionized genomics research, making it difficult to overstate its impact on studies of Biology. NGS will immediately allow researchers working in non‐mainstream species to obtain complete genomes together with a comprehensive catalogue of variants. In addition, RNA‐seq will be a decisive way to annotate genes that cannot be predicted purely by computational or comparative approaches. Future applications include whole genome sequence association studies, as opposed to classical SNP‐based association, and implementing this new source of information into breeding programmes. For these purposes, one of the main advantages of sequencing vs. genotyping is the possibility of identifying copy number variants. Currently, experimental design is a topic of utmost interest, and here we discuss some of the options available, including pools and reduced representation libraries. Although bioinformatics is still an important bottleneck, this limitation is only transient and should not deter animal geneticists from embracing these technologies. 相似文献
15.
16.
The utility of green fluorescent protein in transgenic plants 总被引:30,自引:0,他引:30
Stewart CN 《Plant cell reports》2001,20(5):376-382
The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has proven to be a powerful tool in plant genetic transformation studies. This paper reviews the history and the progression of the expression of GFP variants in transgenic plants. The distinguishing features of the most useful GFPs, such as those including the S65T chromophore mutation and those with dual excitation peaks, are discussed. The review also focuses on the utility of GFP as a visual selectable marker in aiding the plant transformation process; GFP has been more important in monocot transformation compared with dicot transformation. Finally, the potential utility of new fluorescent proteins is speculated upon. 相似文献
17.
《Trends in plant science》2023,28(4):399-414
Phytohormones have crucial roles in plant growth, development, and acclimation to environmental stress; however, measuring phytohormone levels and unraveling their complex signaling networks and interactions remains challenging. Mass spectrometry (MS) has revolutionized the study of complex biological systems, enabling the comprehensive identification and quantification of phytohormones and their related targets. Here, we review recent advances in MS technologies and highlight studies that have used MS to discover and analyze phytohormone-mediated molecular events. In particular, we focus on the application of MS for profiling phytohormones, elucidating phosphorylation signaling, and mapping protein interactions in plants. 相似文献
18.
Confocal fluorescence microscopy of plant cells 总被引:14,自引:0,他引:14
Summary The confocal laser scanning microscope (CLSM) has become a vital instrument for the examination of subcellular structure, especially in fluorescently stained cells. Because of its ability to markedly reduce out-of-focus flare, when compared to the conventional wide-field fluorescence microscope, the CLSM provides a substantial improvement in resolution along the z axis and permits optical sectioning of cells. These developments have been particularly helpful for the investigation of plant cells and tissues, which because of their shape, size, and optical properties have been difficult to analyze at high resolution by conventional means. We review the contribution that the CLSM has made to the study of plant cells. We first consider the principle of operation of the CLSM, including a discussion of image processing, and of lasers and appropriate fluorescent dyes. We then summarize several studies of both fixed and live plant cells in which the instrument has provided new or much clearer information about cellular substructure than has been possible heretofore. Attention is given to the visualization of different components, including especially the cytoskeleton, endomembranes, nuclear components, and relevant ions, and their changes in relationship to physiological and developmental processes. We conclude with an effort to anticipate advances in technology that will improve and extend the performance of the CLSM. In addition to the usual bibliography, we provide internet addresses for information about the CLSM. 相似文献
19.
In the last years, significant advances in microscopy techniques and the introduction of a novel technology to label living
cells with genetically encoded fluorescent proteins revolutionized the field of Cell Biology. Our understanding on cell dynamics
built from snapshots on fixed specimens has evolved thanks to our actual capability to monitor in real time the evolution
of processes in living cells. Among these new tools, single particle tracking techniques were developed to observe and follow
individual particles. Hence, we are starting to unravel the mechanisms driving the motion of a wide variety of cellular components
ranging from organelles to protein molecules by following their way through the cell. In this review, we introduce the single
particle tracking technology to new users. We briefly describe the instrumentation and explain some of the algorithms commonly
used to locate and track particles. Also, we present some common tools used to analyze trajectories and illustrate with some
examples the applications of single particle tracking to study dynamics in living cells. 相似文献
20.
Jeanguenin L Alcon C Duby G Boeglin M Chérel I Gaillard I Zimmermann S Sentenac H Véry AA 《The Plant journal : for cell and molecular biology》2011,67(4):570-582
A functional Shaker potassium channel requires assembly of four α-subunits encoded by a single gene or various genes from the Shaker family. In Arabidopsis thaliana, AtKC1, a Shaker α-subunit that is silent when expressed alone, has been shown to regulate the activity of AKT1 by forming heteromeric AtKC1-AKT1 channels. Here, we investigated whether AtKC1 is a general regulator of channel activity. Co-expression in Xenopus oocytes of a dominant negative (pore-mutated) AtKC1 subunit with the inward Shaker channel subunits KAT1, KAT2 or AKT2, or the outward subunits SKOR or GORK, revealed that the three inward subunits functionally interact with AtKC1 while the outward ones cannot. Localization experiments in plant protoplasts showed that KAT2 was able to re-locate AtKC1 fused to GFP from endomembranes to the plasma membrane, indicating that heteromeric AtKC1-KAT2 channels are efficiently targeted to the plasma membrane. Functional properties of heteromeric channels involving AtKC1 and KAT1, KAT2 or AKT2 were analysed by voltage clamp after co-expression of the respective subunits in Xenopus oocytes. AtKC1 behaved as a regulatory subunit within the heterotetrameric channel, reducing the macroscopic conductance and negatively shifting the channel activation potential. Expression studies showed that AtKC1 and its identified Shaker partners have overlapping expression patterns, supporting the hypothesis of a general regulation of inward channel activity by AtKC1 in planta. Lastly, AtKC1 disruption appeared to reduce plant biomass production, showing that AtKC1-mediated channel activity regulation is required for normal plant growth. 相似文献