scholarly journals A cell-sorting-based nano-scale pipeline for cell-type-specific proteomics in plants

2021 ◽  
Author(s):  
Hongliang Wang ◽  
Rongxia Li ◽  
Jingkai Rong ◽  
Tian Sang ◽  
Chanhong Kim ◽  
...  

Multicellular organisms such as plants contain different types of cells with specialized functions. Analyzing the characteristics of each type of cells will reveal specific cell functions and enhance understanding of how an organism organizes and works. Due to the lack of a method to enrich the particular type of cells and the limited sensitivity and capability of mass spectrometry-based technologies, comprehensive analyses of the global proteome in a single plant cell are still impossible. Here we report a high-sensitive and efficient single-cell-proteomic pipeline, combining simplified flow cytometry-based fluorescent cell-sorting for fluorescent protoplasts and an optimized nano-scale proteomics method, which allows us to identify more than 1,000 unique proteins from 500 guard cell protoplasts. This method was applied to profile the proteome of guard cells and mesophyll cells in Arabidopsis leaves, and epidermal cells in root tips of Arabidopsis and rice seedlings. We also performed an in-depth, quantitative comparison between the proteomics of guard cells and mesophyll cells and revealed that the enrichment of signal transduction-related proteins enables guard cells to respond to various environmental stimuli quickly. This method is applicable to other types of cells in plant or non-plant systems to acquire systemic knowledge of how cells work specifically and in highly organized multiple cell organisms.

Genetics ◽  
2003 ◽  
Vol 163 (4) ◽  
pp. 1427-1438 ◽  
Author(s):  
Mátyás Végh ◽  
Konrad Basler

Abstract The development of multicellular organisms requires the establishment of cell populations with different adhesion properties. In Drosophila, a cell-segregation mechanism underlies the maintenance of the anterior (A) and posterior (P) compartments of the wing imaginal disc. Although engrailed (en) activity contributes to the specification of the differential cell affinity between A and P cells, recent evidence suggests that cell sorting depends largely on the transduction of the Hh signal in A cells. The activator form of Cubitus interruptus (Ci), a transcription factor mediating Hh signaling, defines anterior specificity, indicating that Hh-dependent cell sorting requires Hh target gene expression. However, the identity of the gene(s) contributing to distinct A and P cell affinities is unknown. Here, we report a genetic screen based on the FRT/FLP system to search for genes involved in the correct establishment of the anteroposterior compartment boundary. By using double FRT chromosomes in combination with a wing-specific FLP source we screened 250,000 mutagenized chromosomes. Several complementation groups affecting wing patterning have been isolated, including new alleles of most known Hh-signaling components. Among these, we identified a class of patched (ptc) alleles exhibiting a novel phenotype. These results demonstrate the value of our setup in the identification of genes involved in distinct wing-patterning processes.


Author(s):  
M. Arif Hayat

Although it is recognized that niacin (pyridine-3-carboxylic acid), incorporated as the amide in nicotinamide adenine dinucleotide (NAD) or in nicotinamide adenine dinucleotide phosphate (NADP), is a cofactor in hydrogen transfer in numerous enzyme reactions in all organisms studied, virtually no information is available on the effect of this vitamin on a cell at the submicroscopic level. Since mitochondria act as sites for many hydrogen transfer processes, the possible response of mitochondria to niacin treatment is, therefore, of critical interest.Onion bulbs were placed on vials filled with double distilled water in the dark at 25°C. After two days the bulbs and newly developed root system were transferred to vials containing 0.1% niacin. Root tips were collected at ¼, ½, 1, 2, 4, and 8 hr. intervals after treatment. The tissues were fixed in glutaraldehyde-OsO4 as well as in 2% KMnO4 according to standard procedures. In both cases, the tissues were dehydrated in an acetone series and embedded in Reynolds' lead citrate for 3-10 minutes.


2020 ◽  
Vol 15 (6) ◽  
pp. 531-546 ◽  
Author(s):  
Hwa-Yong Lee ◽  
In-Sun Hong

Recent studies on the mechanisms that link metabolic changes with stem cell fate have deepened our understanding of how specific metabolic pathways can regulate various stem cell functions during the development of an organism. Although it was originally thought to be merely a consequence of the specific cell state, metabolism is currently known to play a critical role in regulating the self-renewal capacity, differentiation potential, and quiescence of stem cells. Many studies in recent years have revealed that metabolic pathways regulate various stem cell behaviors (e.g., selfrenewal, migration, and differentiation) by modulating energy production through glycolysis or oxidative phosphorylation and by regulating the generation of metabolites, which can modulate multiple signaling pathways. Therefore, a more comprehensive understanding of stem cell metabolism could allow us to establish optimal culture conditions and differentiation methods that would increase stem cell expansion and function for cell-based therapies. However, little is known about how metabolic pathways regulate various stem cell functions. In this context, we review the current advances in metabolic research that have revealed functional roles for mitochondrial oxidative phosphorylation, anaerobic glycolysis, and oxidative stress during the self-renewal, differentiation and aging of various adult stem cell types. These approaches could provide novel strategies for the development of metabolic or pharmacological therapies to promote the regenerative potential of stem cells and subsequently promote their therapeutic utility.


Author(s):  
Andrew Clarke

Freezing is a widespread ecological challenge, affecting organisms in over half the terrestrial environment as well as both polar seas. With very few exceptions, if a cell freezes internally, it dies. Polar teleost fish in shallow waters avoid freezing by synthesising a range of protein or glycoprotein antifreezes. Terrestrial organisms are faced with a far greater thermal challenge, and exhibit a more complex array of responses. Unicellular organisms survive freezing temperatures by preventing ice nucleating within the cytosol, and tolerating the cellular dehydration and membrane disruption that follows from ice forming in the external environment. Multicellular organisms survive freezing temperatures by manipulating the composition of the extracellular body fluids. Terrestrial organisms may freeze at high subzero temperatures, often promoted by ice nucleating proteins, and small molecular mass cryoprotectants (often sugars and polyols) moderate the osmotic stress on cells. A range of chaperone proteins (dehydrins, LEA proteins) help maintain the integrity of membranes and macromolecules. Thermal hysteresis (antifreeze) proteins prevent damaging recrystallisation of ice. In some cases arthropods and higher plants prevent freezing in their extracellular fluids and survive by supercooling. Vitrification of extracellular water, or of the cell cytosol, may be a more widespread response to very cold temperatures than recognised to date.


2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jean Chemin ◽  
Tamara Timic Stamenic ◽  
Magalie Cazade ◽  
Jodie Llinares ◽  
Iulia Blesneac ◽  
...  

Abstract Cav3 / T-type Ca2+ channels are dynamically regulated by intracellular Ca2+ ions, which inhibit Cav3 availability. Here, we demonstrate that this inhibition becomes irreversible in the presence of non-hydrolysable ATP analogs, resulting in a strong hyperpolarizing shift in the steady-state inactivation of the residual Cav3 current. Importantly, the effect of these ATP analogs was prevented in the presence of intracellular BAPTA. Additional findings obtained using intracellular dialysis of inorganic phosphate and alkaline phosphatase or NaN3 treatment further support the involvement of a phosphorylation mechanism. Contrasting with Cav1 and Cav2 Ca2+ channels, the Ca2+-dependent modulation of Cav3 channels appears to be independent of calmodulin, calcineurin and endocytic pathways. Similar findings were obtained for the native T-type Ca2+ current recorded in rat thalamic neurons of the central medial nucleus. Overall, our data reveal a new Ca2+ sensitive phosphorylation-dependent mechanism regulating Cav3 channels, with potentially important physiological implications for the multiple cell functions controlled by T-type Ca2+ channels.


Cancers ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1299
Author(s):  
Yi Wu ◽  
Peng Huang ◽  
Xian-Ping Dong

Ca2+ is pivotal intracellular messenger that coordinates multiple cell functions such as fertilization, growth, differentiation, and viability. Intracellular Ca2+ signaling is regulated by both extracellular Ca2+ entry and Ca2+ release from intracellular stores. Apart from working as the cellular recycling center, the lysosome has been increasingly recognized as a significant intracellular Ca2+ store that provides Ca2+ to regulate many cellular processes. The lysosome also talks to other organelles by releasing and taking up Ca2+. In lysosomal Ca2+-dependent processes, autophagy is particularly important, because it has been implicated in many human diseases including cancer. This review will discuss the major components of lysosomal Ca2+ stores and their roles in autophagy and human cancer progression.


2012 ◽  
Vol 153 (6) ◽  
pp. 214-221 ◽  
Author(s):  
Veronika Urbán S. ◽  
Elizabeta Benevolenskaya ◽  
Judit Kiss ◽  
Bernadett Sági ◽  
Beáta Hegyi ◽  
...  

Analysis of genomic sequences has clearly shown that the genomic differences among species do not explain the diversity of life. The genetic code itself serves as only a part of the dynamic complexity that results in the temporal and spatial changes in cell phenotypes during development. It has been concluded that the phenotype of a cell and of the organism as a whole is more influenced by environmentally-induced changes in gene activity than had been previously thought. The emerging field of epigenetics focuses on molecular marks on chromatin; called the epigenome, which serve as transmitters between the genome and the environment. These changes not only persist through multiple cell division cycles, but may also endure for multiple generations. Irregular alterations of the epigenome; called epimutations, may have a decisive role in the etiology of human pathologies such as malignancies and other complex human diseases. Epigenetics can provide the missing link between genetics, disease and the environment. Therefore, this field may have an increasing impact on future drug design and serve as a basis for new therapeutic/preventative approaches. Orv. Hetil., 2012, 153, 214–221.


2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Veenstra TD ◽  

Identifying all the molecular components within a living cell is the first step into understanding how it functions. To further understand how a cell functions requires identifying the interactions that occur between these components. This fact is especially relevant for proteins. No protein within a human cell functions on its own without interacting with another biomolecule - usually another protein. While Protein-Protein Interactions (PPI) have historically been determined by examining a single protein per study, novel technologies developed over the past couple of decades are enabling high-throughput methods that aim to describe entire protein networks within cells. In this review, some of the technologies that have led to these developments are described along with applications of these techniques. Ultimately the goal of these technologies is to map out the entire circuitry of PPI within human cells to be able to predict the global consequences of perturbations to the cell system. This predictive capability will have major impacts on the future of both disease diagnosis and treatment.


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