RESUMO
The field of plant cell biology has a rich history of discovery, going back to Robert Hooke's discovery of cells themselves. The development of microscopes and preparation techniques has allowed for the visualization of subcellular structures, and the use of protein biochemistry, genetics, and molecular biology has enabled the identification of proteins and mechanisms that regulate key cellular processes. In this review, seven senior plant cell biologists reflect on the development of this research field in the past decades, including the foundational contributions that their teams have made to our rich, current insights into cell biology. Topics covered include signaling and cell morphogenesis, membrane trafficking, cytokinesis, cytoskeletal regulation, and cell wall biology. In addition, these scientists illustrate the pathways to discovery in this exciting research field.
Assuntos
Parede Celular , Citocinese , Citoesqueleto , Células Vegetais , Fenômenos Fisiológicos Vegetais , Transdução de Sinais , Biologia CelularRESUMO
Understanding the changes in diverse molecular pathways underlying the development of breast tumors is critical for improving diagnosis, treatment, and drug development. Here, we used RNA-profiling of canine mammary tumors (CMTs) coupled with a robust analysis framework to model molecular changes in human breast cancer. Our study leveraged a key advantage of the canine model, the frequent presence of multiple naturally occurring tumors at diagnosis, thus providing samples spanning normal tissue and benign and malignant tumors from each patient. We showed human breast cancer signals, at both expression and mutation level, are evident in CMTs. Profiling multiple tumors per patient enabled by the CMT model allowed us to resolve statistically robust transcription patterns and biological pathways specific to malignant tumors versus those arising in benign tumors or shared with normal tissues. We showed that multiple histological samples per patient is necessary to effectively capture these progression-related signatures, and that carcinoma-specific signatures are predictive of survival for human breast cancer patients. To catalyze and support similar analyses and use of the CMT model by other biomedical researchers, we provide FREYA, a robust data processing pipeline and statistical analyses framework.
RESUMO
The false discovery rate (FDR) measures the proportion of false discoveries among a set of hypothesis tests called significant. This quantity is typically estimated based on p-values or test statistics. In some scenarios, there is additional information available that may be used to more accurately estimate the FDR. We develop a new framework for formulating and estimating FDRs and q-values when an additional piece of information, which we call an "informative variable", is available. For a given test, the informative variable provides information about the prior probability a null hypothesis is true or the power of that particular test. The FDR is then treated as a function of this informative variable. We consider two applications in genomics. Our first application is a genetics of gene expression (eQTL) experiment in yeast where every genetic marker and gene expression trait pair are tested for associations. The informative variable in this case is the distance between each genetic marker and gene. Our second application is to detect differentially expressed genes in an RNA-seq study carried out in mice. The informative variable in this study is the per-gene read depth. The framework we develop is quite general, and it should be useful in a broad range of scientific applications.
Assuntos
Marcadores Genéticos , Genômica , Animais , Perfilação da Expressão Gênica , Marcadores Genéticos/genética , Genômica/métodos , Camundongos , Probabilidade , Saccharomyces cerevisiae/genéticaRESUMO
It has been proposed by some plant scientists that plants are cognitive and conscious organisms, although this is a minority view. Here we present a brief summary of some of the arguments against this view, followed by a critique of an article in this same issue of Biochemical and Biophysical Research Communications by Calvo, Baluska, and Trewavas (2020) that cites Integrated Information Theory (IIT) as providing additional support for plant consciousness. The authors base their argument on the assumptions that all cells are conscious and that consciousness is confined to life. However, IIT allows for consciousness in various nonliving systems, and thus does not restrict consciousness to living organisms. Therefore, IIT cannot be used to prove plant consciousness, for which there is neither empirical evidence nor support from other, neuron-based, theories of consciousness.
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Estado de Consciência/fisiologia , Teoria da Informação , Plantas/metabolismo , HumanosRESUMO
The plant Golgi apparatus (sensu lato: Golgi stack + Trans Golgi Network, TGN) is a highly polar and mobile key organelle lying at the junction of the secretory and endocytic pathways. Unlike its counterpart in animal cells it does not disassemble during mitosis. It modifies glycoproteins sent to it from the endoplasmic reticulum (ER), it recycles ER resident proteins, it sorts proteins destined for the vacuole from secretory proteins, it receives proteins internalised from the plasma membrane and either recycles them to the plasma membrane or retargets them to the vacuole for degradation. In functional terms the Golgi apparatus can be likened to a car factory, with incoming (COPII traffic) and returning (COPI traffic) railway lines at the entry gate, and a distribution centre (the TGN) at the exit gate of the assembly hall. In the assembly hall we have a conveyor belt system where the incoming car parts are initially assembled (in the cis-area) then gradually modified into different models (processing of secretory cargo) as the cars pass along the production line (cisternal maturation). After being released the trans-area, the cars (secretory cargos) are moved out of the assembly hall and passed on to the distribution centre (TGN), where the various models are placed onto different trains (cargo sorting into carrier vesicles) for transport to the car dealers. Cars with motor problems are returned to the factory for repairs (endocytosis to the TGN). This simple analogy also incorporates features of quality control at the COPII entry gate with defective parts being returned to the manufacturing center (the ER) via the COPI trains (vesicles). In recent years, numerous studies have contributed to our knowledge on Golgi function and structure in both animals, yeast and plants. This review, rather than giving a balanced account of the structure as well as of the function of the Golgi apparatus has purposely a marked slant towards plant Golgi ultrastructure integrating findings from the mammalian/animal field.
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Complexo de Golgi/ultraestrutura , Células Vegetais/ultraestrutura , Vesículas Revestidas/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Microscopia Eletrônica , Vesículas Secretórias/ultraestrutura , Vesículas Transportadoras/ultraestrutura , Rede trans-Golgi/ultraestruturaRESUMO
BACKGROUND: Quiescence, reversible exit from the cell division cycle, is characterized by large-scale changes in steady-state gene expression, yet mechanisms controlling these changes are in need of further elucidation. In order to characterize the effects of post-transcriptional control on the quiescent transcriptome in human fibroblasts, we determined mRNA decay rates for over 10,000 genes using a transcription shut-off time-course. RESULTS: We found that ~500 of the genes monitored exhibited significant changes in decay rate upon quiescence induction. Genes involved in RNA processing and ribosome biogenesis were destabilized with quiescence, while genes involved in the developmental process were stabilized with quiescence. Moreover, extracellular matrix genes demonstrated an upregulation of gene expression that corresponded with a stabilization of these transcripts. Additionally, targets of a quiescence-associated microRNA (miR-29) were significantly enriched in the fraction of transcripts that were stabilized during quiescence. CONCLUSION: Coordinated stability changes in clusters of genes with important functions in fibroblast quiescence maintenance are highly correlated with quiescence gene expression patterns. Analysis of miR-29 target decay rates suggests that microRNA-induced changes in RNA stability are important contributors to the quiescence gene expression program in fibroblasts. The identification of multiple stability-related gene clusters suggests that other posttranscriptional regulators of transcript stability may contribute to the coordination of quiescence gene expression. Such regulators may ultimately prove to be valuable targets for therapeutics that target proliferative cells, for instance, in cancer or fibrosis.
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Fibroblastos/metabolismo , Regulação da Expressão Gênica , Estabilidade de RNA , RNA Mensageiro/genética , Fase de Repouso do Ciclo Celular/genética , Análise por Conglomerados , Perfilação da Expressão Gênica , Genoma Humano , Estudo de Associação Genômica Ampla , Genômica/métodos , Humanos , Interferência de RNA , TranscriptomaRESUMO
Tissue morphogenesis in plants requires communication between cells, a process involving the trafficking of molecules through plasmodesmata (PD). PD conductivity is regulated by endogenous and exogenous signals. However, the underlying signaling mechanisms remain enigmatic. In Arabidopsis, signal transduction mediated by the receptor-like kinase STRUBBELIG (SUB) contributes to inter-cell layer signaling during tissue morphogenesis. Previous analysis has revealed that SUB acts non-cell-autonomously suggesting that SUB controls tissue morphogenesis by participating in the formation or propagation of a downstream mobile signal. A genetic screen identified QUIRKY (QKY), encoding a predicted membrane-anchored C2-domain protein, as a component of SUB signaling. Here, we provide further insight into the role of QKY in this process. We show that like SUB, QKY exhibits non-cell-autonomy when expressed in a tissue-specific manner and that non-autonomy of QKY extends across several cells. In addition, we report on localization studies indicating that QKY and SUB localize to PD but independently of each other. FRET-FLIM analysis suggests that SUB and QKY are in close contact at PD in vivo. We propose a model where SUB and QKY interact at PD to promote tissue morphogenesis, thereby linking RLK-dependent signal transduction and intercellular communication mediated by PD.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Plasmodesmos/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Plasmodesmos/genética , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais/fisiologiaRESUMO
Understanding the differences between microarray and RNA-Seq technologies for measuring gene expression is necessary for informed design of experiments and choice of data analysis methods. Previous comparisons have come to sometimes contradictory conclusions, which we suggest result from a lack of attention to the intensity-dependent nature of variation generated by the technologies. To examine this trend, we carried out a parallel nested experiment performed simultaneously on the two technologies that systematically split variation into four stages (treatment, biological variation, library preparation and chip/lane noise), allowing a separation and comparison of the sources of variation in a well-controlled cellular system, Saccharomyces cerevisiae. With this novel dataset, we demonstrate that power and accuracy are more dependent on per-gene read depth in RNA-Seq than they are on fluorescence intensity in microarrays. However, we carried out quantitative PCR validations which indicate that microarrays may demonstrate greater systematic bias in low-intensity genes than in RNA-seq.
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Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência de RNA/métodos , Interpretação Estatística de Dados , Genes Fúngicos , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/genéticaRESUMO
The endoplasmic reticulum (ER) is the gateway to the secretory pathway in all eukaryotic cells. Its products subsequently pass through the Golgi apparatus on the way to the cell surface (true secretion) or to the lytic compartment of the cell (vacuolar protein transport). In animal cells, the Golgi apparatus is present as a stationary larger order complex near the nucleus, and transport between the cortical ER and the Golgi complex occurs via an intermediate compartment which is transported on microtubules. By contrast, higher plant cells have discrete mobile Golgi stacks that move along the cortical ER, and the intermediate compartment is absent. Although many of the major molecular players involved in ER-Golgi trafficking in mammalian and yeast (Saccharomyces cerevisiae) cells have homologs in higher plants, the narrow interface (less than 500 nm) between the Golgi and the ER, together with the motility factor, makes the identification of the transport vectors responsible for bidirectional traffic between these two organelles much more difficult. Over the years, a controversy has arisen over the two major possibilities by which transfer can occur: through vesicles or direct tubular connections. In this article, four leading plant cell biologists attempted to resolve this issue. Unfortunately, their opinions are so divergent and often opposing that it was not possible to reach a consensus. Thus, we decided to let each tell his or her version individually. The review begins with an article by Federica Brandizzi that provides the necessary molecular background on coat protein complexes in relation to the so-called secretory units model for ER-Golgi transport in highly vacuolated plant cells. The second article, written by Chris Hawes, presents the evidence in favor of tubules. It is followed by an article from David Robinson defending the classical notion that transport occurs via vesicles. The last article, by Akihiko Nakano, introduces the reader to possible alternatives to vesicles or tubules, which are now emerging as a result of exciting new developments in high-resolution light microscopy in yeast.
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Retículo Endoplasmático/metabolismo , Eucariotos/metabolismo , Complexo de Golgi/metabolismo , Plantas/metabolismo , Transporte Biológico , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/ultraestrutura , Modelos BiológicosRESUMO
Autophagosomes are organelles that deliver cytosolic proteins for degradation in the vacuole of the cell. In contrast, exocyst-positive organelles (EXPO) deliver cytosolic proteins to the cell surface and therefore represent a form of unconventional protein secretion. Because both structures have two boundary membranes, it has been suggested that they may have been falsely treated as separate entities. Using suspension culture cells and root tissue cells of transgenic Arabidopsis (Arabidopsis thaliana) plants expressing either the EXPO marker Arabidopsis Exo70E2-GFP or the autophagosome marker yellow fluorescent protein (YFP)-autophagy-related gene 8e/f (ATG8e/f), and using specific antibodies against Exo70E2 and ATG8, we have now established that, in normally growing cells, EXPO and autophagosomes are distinct from one another. However, when cells/roots are subjected to autophagy induction, EXPO as well as autophagosomes fuse with the vacuole. In the presence of concanamycin A, the punctate fluorescent signals from both organelles inside the vacuole remain visible for hours and overlap to a significant degree. Tonoplast staining with FM4-64/YFP-Rab7-like GTPase/YFP-vesicle-associated membrane protein711 confirmed the internalization of tonoplast membrane concomitant with the sequestration of EXPO and autophagosomes. This suggests that EXPO and autophagosomes may be related to one another; however, whereas induction of autophagy led to an increase in the amount of ATG8 recruited to membranes, Exo70E2 did not respond in a similar manner.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fagossomos/metabolismo , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Autofagia , Família da Proteína 8 Relacionada à Autofagia , Citoplasma/metabolismo , Proteínas de Fluorescência Verde , Proteínas Associadas aos Microtúbulos/genética , Raízes de Plantas/citologia , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas de Transporte Vesicular/genéticaRESUMO
To prevent their being released to the cell exterior, acid hydrolases are recognized by receptors at some point in the secretory pathway and diverted towards the lytic compartment of the cell (lysosome or vacuole). In animal cells, the receptor is called the mannosyl 6-phosphate receptor (MPR) and it binds hydrolase ligands in the trans-Golgi network (TGN). These ligands are then sequestered into clathrin-coated vesicles (CCVs) because of motifs in the cytosolic tail of the MPR which interact first with monomeric adaptors (Golgi-localized, Gamma-ear-containing, ARF-binding proteins, GGAs) and then with tetrameric (adaptin) adaptor complexes. The CCVs then fuse with an early endosome, whose more acidic lumen causes the ligands to dissociate. The MPRs are then recycled back to the TGN via retromer-coated carriers. Plants have vacuolar sorting receptors (VSRs) which were originally identified in CCVs isolated from pea (Pisum sativum L.) cotyledons. It was therefore assumed that VSRs would have an analogous function in plants to MPRs in animals. Although this dogma has enjoyed wide support over the last 20 years there are many inconsistencies. Recently, results have been published which are quite contrary to it. It now emerges that VSRs and their ligands can interact very early in the secretory pathway, and dissociate in the TGN, which, in contrast to its mammalian counterpart, has a pH of 5.5. Multivesicular endosomes in plants lack proton pump complexes and consequently have an almost neutral internal pH, which discounts them as organelles of pH-dependent receptor-ligand dissociation. These data force a critical re-evaluation of the role of CCVs at the TGN, especially considering that vacuolar cargo ligands have never been identified in them. We propose that one population of TGN-derived CCVs participate in retrograde transport of VSRs from the TGN. We also present a new model to explain how secretory and vacuolar cargo proteins are effectively separated after entering the late Golgi/TGN compartments.
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Proteínas de Transporte/metabolismo , Receptores de Superfície Celular/metabolismo , Vacúolos/metabolismo , Animais , Vesículas Revestidas por Clatrina/metabolismo , Vesículas Revestidas/metabolismo , Complexo de Golgi/metabolismo , Modelos Biológicos , Plantas/metabolismo , Rede trans-Golgi/metabolismoRESUMO
The p24 proteins belong to a family of type I membrane proteins which cycle between the endoplasmic reticulum (ER) and Golgi via coat protein I (COPI) and COPII vesicles. Current nomenclature classifies them into four subfamilies, although plant p24 proteins belong to either the p24ß or the p24δ subfamilies. Here, we show that Arabidopsis p24δ5/δ9 and HDEL ligands shift the steady-state distribution of the K/HDEL receptor ERD2 from the Golgi to the ER. We also show that p24δ5/δ9 interact directly with ERD2. This interaction requires the Golgi dynamics (GOLD) domain in p24δ5 and is much higher at acidic than at neutral pH, consistent with both proteins interacting at the cis-Golgi. In addition, p24δ5 also inhibits the secretion of HDEL ligands, but not constitutive secretion, showing a role for p24δ5 in retrograde Golgi-to-ER transport. Both p24δ5 and ERD2 interact with ADP-ribosylation factor 1 (ARF1) and COPI subunits, mostly at acidic pH, consistent with COPI vesicles being involved in retrograde transport of both proteins. In contrast, both proteins interact with the COPII subunit Sec23, mostly at neutral pH, consistent with this interaction taking place at the ER for anterograde transport to the Golgi apparatus.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Peptídeos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Complexo I de Proteína do Envoltório/genética , Complexo I de Proteína do Envoltório/metabolismo , Proteínas de Ligação a DNA/genética , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/genética , Transporte Proteico , Receptores de Peptídeos/genética , Fatores de Transcrição/genéticaRESUMO
MOTIVATION: Next-generation sequencing experiments, such as RNA-Seq, play an increasingly important role in biological research. One complication is that the power and accuracy of such experiments depend substantially on the number of reads sequenced, so it is important and challenging to determine the optimal read depth for an experiment or to verify whether one has adequate depth in an existing experiment. RESULTS: By randomly sampling lower depths from a sequencing experiment and determining where the saturation of power and accuracy occurs, one can determine what the most useful depth should be for future experiments, and furthermore, confirm whether an existing experiment had sufficient depth to justify its conclusions. We introduce the subSeq R package, which uses a novel efficient approach to perform this subsampling and to calculate informative metrics at each depth. AVAILABILITY AND IMPLEMENTATION: The subSeq R package is available at http://github.com/StoreyLab/subSeq/.
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Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de RNA/métodos , Software , Animais , RatosRESUMO
Transient gene expression, in plant protoplasts or specific plant tissues, is a key technique in plant molecular cell biology, aimed at exploring gene products and their modifications to examine functional subdomains, their interactions with other biomolecules, and their subcellular localization. Here, we highlight some of the major advantages and potential pitfalls of the most commonly used transient gene expression models and illustrate how ectopic expression and the use of dominant mutants can provide insights into protein function.
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Pesquisa , Via Secretória , Transporte Biológico , Corantes Fluorescentes/metabolismo , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Protoplastos/metabolismo , Protoplastos/ultraestruturaRESUMO
ADP-ribosylation factor1 (Arf1), a member of the small GTP-binding proteins, plays a pivotal role in protein trafficking to multiple organelles. In its GDP-bound form, Arf1 is recruited from the cytosol to organelle membranes, where it functions in vesicle-mediated protein trafficking. However, the mechanism of Arf1-GDP recruitment remains unknown. Here, we provide evidence that two Glo3p-type Arf GTPase-activating proteins (ArfGAPs), ArfGAP domain8 (AGD8) and AGD9, are involved in the recruitment of Arf1-GDP to the Golgi apparatus in Arabidopsis (Arabidopsis thaliana). RNA interference plants expressing low levels of AGD8 and AGD9 exhibited abnormal Golgi morphology, inhibition of protein trafficking, and arrest of plant growth and development. In RNA interference plants, Arf1 was poorly recruited to the Golgi apparatus. Conversely, high levels of AGD8 and AGD9 induced Arf1 accumulation at the Golgi and suppressed Golgi disruption and inhibition of vacuolar trafficking that was caused by overexpression of AGD7. Based on these results, we propose that the Glo3p-type ArfGAPs AGD8 and AGD9 recruit Arf1-GDP from the cytosol to the Golgi for Arf1-mediated protein trafficking, which is essential for plant development and growth.