Purification Of Organelles Research Articles

Organelle biogenesis and communication in plant cells

What a wonderful time this is for plant cell biology! Never before has a wealth of tools been adopted and adapted to dissect the molecular mechanisms underlying the functions of the plant cell. Genome sequencing, subcellular proteomics, and advanced imaging are just some of the astounding array of technologies that hold the promise to revolutionize the way we can study and appreciate plant cells. In a nutshell, this is what this Special Issue of Plant Cell Reports on ‘Organelle Biogenesis and Communication in Plant Cell’—the first of its kind for this journal—aims to bring to light in a unique collection of reviews and research articles. With no doubt, recent advances in organelle purification and proteomics coupled with entire genome sequencing are yielding important discoveries on species evolution as highlighted by Jo et al. (2011) and Lang et al. (2011), and advanced microscopy is allowing for the visualization and tracking of previously unknown aspects of organelle dynamics at a subcellular level in intact tissues, such as the regulation of mitochondrial morphology and motility in embryos presented by Yamaoka et al. (2011). Indeed, it is now possible to understand how organelles are implicated in more general aspects of plant biology, including cytoplasmic male sterility, as discussed by Matsunaga et al. (2011), nitrogen assimilation, as reviewed by Hakiya et al. (2011), and the regulation of developmental pathways, which, as reviewed by Merkle (2011), can be controlled through the nucleo-cytoplasmic transport of their signaling components. Finally, there have also been unprecedented advances in our understanding of the role of lipids and proteins in organelle membrane assembly, aspects of which are reviewed by Melser et al. (2011) and Abell and Mullen (2011). Taken together, the reviews and articles in this Special Issue of Plant Cell Reports summarize some of the stateof-the-art research in the field of plant cell biology and how our view of the cell is being constantly challenged as new information is acquired. However, this is only a facet of plant cell biology and the reader is encouraged to examine future issues of Plant Cell Reports for further examples of research that highlight this exciting field. We are indebted to all of the contributing authors for their thoughtprovoking reviews and research articles and to those colleagues that acted as reviewers.

Molecular characterization of the endoplasmic reticulum: Insights from proteomic studies

The endoplasmic reticulum (ER) is a multifunctional intracellular organelle responsible for the synthesis, processing and trafficking of a wide variety of proteins essential for cell growth and survival. Therefore, comprehensive characterization of the ER proteome is of great importance to the understanding of its functions and has been actively pursued in the past decade by scientists in the proteomics field. This review summarizes major proteomic studies published in the past decade that focused on the ER proteome. We evaluate the data sets obtained from two different organs, liver and pancreas each of which contains a primary cell type (hepatocyte and acinar cell) with specialized functions. We also discuss how the nature of the proteins uncovered is related to the methods of organelle purification, organelle purity and the techniques used for protein separation prior to MS. In addition, this review also puts emphasis on the biological insights gained from these studies regarding the molecular functions of the ER including protein synthesis and translocation, protein folding and quality control, ER-associated degradation and ER stress, ER export and membrane trafficking, calcium homeostasis and detoxification and drug metabolism.

8-Parallel Bioparticle Sorter with a Multilayer PDMS Chip

A major challenge in organelle proteomics is purifying the organelle of interest from cell lysate. We have been developing a fluorescence-activated microfluidic sorter for organelle purification. Flow switching mechanism of the sorter is based on the sol-gel transition of a thermoreversible gelation polymer (TGP). The TGP is liquid at low temperature and turns to a gel immediately upon heating. The TGP solution is mixed with bioparticles and introduced into a mirochannel that branches into waste and collection channels. When the fluorescence signal from a target particle is detected, the sol-gel transition of the TGP is induced at the entrance of the waste channel by heating with an infrared laser. This operation allows a fluorescent target to flow into the collection channel. In this study, eight parallel sorters were integrated in a microchip to achieve high-throughput sorting. The microchip consists of a glass cover plate and a PDMS block. The PDMS block has a multilayer structure with two inlets for sample and buffer solutions and two outlets for collection and waste solutions. Its structure makes it possible to form a sheath flow, i.e., sample-solution flow is focused by neighboring two buffer-solution flows in each sorter. We confirmed that sheath flows were formed in the chip. We demonstrated that fluorescent microspheres and E. coli cells were detected and sorted in each channel. A recovery ratio of 90% and a throughput of 20 particles/s were achieved. Future studies will allow us to integrate 16 or 32 channels into a single chip. The parallel sorter is a promising device for purifying large amount of bioparticles.

Sub-cellular distribution of glutathione in an Arabidopsis mutant ( vtc1) deficient in ascorbate

Glutathione and ascorbate are considered the major redox buffers in plant cells. They are implicated in many reactions at the sub-cellular level. However, information about the location and quantification of glutathione in the different sub-cellular compartments is very scarce and it has been obtained mainly using organelle purification and chemical quantification. We have used a glutathione antibody to immunolabel and quantify the total glutathione in leaves from wild-type Arabidopsis thaliana (Col-0) and an A. thaliana mutant (vtc1) deficient in ascorbate. Spectrophotometrical quantification has shown that this mutant has a higher content of glutathione during plant development compared with Col-0 [Pavet V, Olmos E, Kiddle G, Mowla S, Kumar S, Antoniw J, et al. Ascorbic acid deficiency activates cell death and disease resistance responses in Arabidopsis. Plant Physiology 2005;139:1291-03]. We have observed, using immunolabelling techniques, that mitochondria showed the highest density of glutathione labelling in both Col-0 and vtc1 plants during all developmental stages and that the lowest density occurred in the chloroplasts, for both lines. However, the distribution of glutathione in the different sub-cellular compartments indicates that the chloroplasts contain about 62-75% of the total cellular glutathione and that the mitochondria represent the second greatest pool, with about 15-25% of the total cellular glutathione. It has been observed previously that the vtc1 mutant exhibits an induction of cell death and disease resistance in the face of pathogen attack. The differing distributions and concentrations of glutathione in the mitochondria of wild-type A. thaliana and the vtc1 mutant is discussed.

A Quantitative Proteomic Analysis of Mitochondrial Participation in P19 Cell Neuronal Differentiation

A quantitative proteomic analysis of changes in protein expression accompanying the differentiation of P19 mouse embryonal carcinoma cells into neuron-like cells using isobaric tag technology coupled with LC-MS/MS revealed protein changes reflecting withdrawal from the cell cycle accompanied by a dynamic reorganization of the cytoskeleton and an up-regulation of mitochondrial biogenesis. Further study of quantitative changes in abundance of individual proteins in a purified mitochondrial fraction showed that most mitochondrial proteins increased significantly in abundance. A set of chaperone proteins did not participate in this increase, suggesting that neuron-like cells are relatively deficient in mitochondrial chaperones. We developed a procedure to account for differences in recovery of mitochondrial proteins during purification of organelles from distinct cell or tissue sources. Proteomic data supported by RT-PCR analysis suggests that enhanced mitochondrial biogenesis during neuronal differentiation may reflect a large increase in expression of PGC-1alpha combined with down-regulation of its negative regulator, p160 Mybbp1a.

The nuclear proteome and DNA-binding fraction of human Raji lymphoma cells

Purification of organelles and analysis of their proteins is an important initial step for biological proteomics, simplifying the proteome prior to analysis by established techniques such as two-dimensional liquid chromatography (2-DLC) or two-dimensional gel electrophoresis (2-DE). Nuclear proteins play a central role in regulating gene expression, but are often under-represented in proteomic studies due to their lower abundance in comparison to cellular ‘housekeeping’ metabolic enzymes and structural proteins. A reliable procedure for separation and proteomic analysis of nuclear proteins would be useful for investigations of cell proliferation and differentiation during disease processes (e.g., human cancer). In this study, we have purified nuclei from the human Burkitt's lymphoma B-cell line, Raji, using sucrose density gradient centrifugation. The integrity and purity of the nuclei were assessed by light microscopy and proteins from the nuclear fractions were separated by 2-DE and identified using matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). A total of 124 unique proteins were identified, of which 91% ( n = 110) were predicted to be nuclear using PSORT. Proteins from the nuclear fraction were subjected to affinity chromatography on DNA-agarose to isolate DNA-binding proteins. From this purified fraction, 131 unique proteins were identified, of which 69% ( n = 90) were known or predicted DNA-binding proteins. Purification of nuclei and subsequent enrichment of DNA-binding proteins allowed identification of a total of 209 unique proteins, many involved in transcription and/or correlated with lymphoma, leukemia or cancer in general. The data obtained should be valuable for identification of biomarkers and targets for cancer therapy, and for furthering our understanding of the molecular mechanisms underlying lymphoma development and progression.

Zebrafish as a Novel Model System to Study the Function of Caveolae and Caveolin-1 in Organismal Biology

Caveolin-1 (Cav-1) was first identified as a marker protein for the purification of caveolae organelles.1–3 Subsequently, it was later determined that Cav-1 expression is essential for caveolae formation.4,5 Thus, Cav-1(−/−)-deficient mice morphologically lack caveolae organelles. Surprisingly, these mice are viable and fertile.4,5 In striking contrast, zebrafish (Danio rerio) lacking Cav-1 display important developmental abnormalities and embryonic lethality. These novel findings by Fang et al are described and highlighted in this issue of The American Journal of Pathology.6

High‐throughput localization of organelle proteins by mass spectrometry: a quantum leap for cell biology

Cells are the fundamental building blocks of organisms and their organization holds the key to our understanding of the processes that control Development and Physiology as well as the mechanisms that underlie disease. Traditional methods of analysis of subcellular structure have relied on the purification of organelles and the painstaking biochemical description of their components. The arrival of high-throughput genomic and, more significantly, proteomic technologies has opened hereto unforeseen possibilities for this task. Recently two reports((1,2)) show how much can be gleaned from the combination of analytical centrifugation, mass spectrometry and advanced statistical techniques focused on a high-throughput analysis of the content and organization of plant and animal cells. The results reveal intriguing possibilities for the future and the possibility of mapping much of the known proteome onto our current map of the cell.

Proteomics of synapse

Large-scale phosphoproteome analysis on synaptosome and preparation of post-synaptic density (PSD) were investigated. It was found that protein phosphor is a common event in the synapse, which is consistent with the presence of diverse classes of kinases and phosphatases in the synapse. Synaptic proteomics analysis required the purification of subcellular organelles from the brain regions of interest. Multiple steps of discontinuous density gradient ultra-centrifugation were employed to enrich the distinct organelles. Two-dimensional gel electrophoresis was used to separate and quantify proteins, including post-translational modified forms, from synaptic structures. It was observed that proteomic analysis of the synaptic vesicle identified 36 proteins, including seven integral membrane proteins and vesicle regulatory proteins.

Purification of Organelles from Mammalian Cells

The protocols in this unit illustrate a range of different procedures that have been used to fractionate tissue homogenates. They emphasize different fractionation techniques that have been used for rat liver, an abundant tissue that has been a favorite of many investigators and has served as the source of many organelle preparations of excellent purity. For selected procedures, other examples have been given using other tissue sources (e.g., glandular tissues that maintain protein storage granules for regulated secretion) or, where particularly favorable, cultured cells. The basis (or goal) of each separation and the merits and limitations of each procedure are summarized to provide a guide for selecting among the various approaches. The large number of protocols include specific details relevant to a particular sample cell type (and in many cases a particular organelle).

Purification of Organelles from Mammalian Cells

Organelle purification procedures capitalize on the differences in size, density, and (occasionally) surface charge density of individual types of organelles. Most fractionation procedures that are based on centrifugation involve some combination of procedures that distinguish both size and density. Initially, a homogenate is prepared in isoosmotic (or slightly hyperosmotic) sucrose or some other predominantly nonelectrolyte medium. A wide range of procedures have been used to fractionate tissue homogenates. The protocols in this unit emphasize different fractionation techniques that have been used for rat liver, an abundant tissue that has been a favorite of many investigators and has served as the source of many organelle preparations of excellent purity. For selected procedures, examples have been given using other tissue sources (e.g., glandular tissues that maintain protein storage granules for regulated secretion) or, where particularly favorable, cultured cells.

Proteomic Consequences of a Human Mitochondrial tRNA Mutation beyond the Frame of Mitochondrial Translation

Numerous severe neurodegenerative and neuromuscular disorders, characterized biochemically by strong perturbations in energy metabolism, are correlated with single point mutations in mitochondrial genes coding for transfer RNAs. Initial comparative proteomics performed on wild-type and Myoclonic Epilepsy and Ragged Red Fibers (MERRF) mitochondria from sibling human cybrid cell lines revealed the potential of this approach. Here a quantitative analysis of several hundred silver-stained spots separated by two-dimensional gel electrophoresis was performed in the specific case of a couple of mitochondria, containing or not mutation A8344G in the gene for mitochondrial tRNALys, correlated with MERRF syndrome. Computer-assisted analysis allowed us to detect 38 spots with significant quantitative variations, of which 20 could be assigned by mass spectrometry. These include nuclear encoded proteins located in mitochondria such as respiratory chain subunits, metabolic enzymes, a protein of the mitochondrial translation machinery, and cytosolic contaminants. Furthermore, Western blotting combined with mass spectrometry revealed the occurrence of numerous isoforms of pyruvate dehydrogenase subunits, with subtle changes in post-translational modifications. This comparative proteomic approach gives the first insight for nuclear encoded proteins that undergo the largest quantitative changes, and pinpoints new potential molecular partners involved in the cascade of events that connect genotype to phenotype.

Isolation of organelles and prefractionation of protein extracts using free-flow electrophoresis.

One of the major obstacles in the analysis of proteomes is the extreme complexity of any particular cell or biological fluid. Free-flow electrophoresis (FFE) is a powerful tool for reduction of this complexity, which is a prerequisite for systematic and comprehensive protein analyses. Protocols are provided in this unit for sample fractionation at two different stages: on the protein level by isoelectric focusing FFE fractionation of crude protein mixtures such as whole cell lysates, and on a subcellular level by zone-electrophoretic FFE purification of organelles.

Organelle purification and selective permeabilisation of the plasma membrane: two different approaches to study vacuoles of the filamentous fungus Ashbya gossypii

Two different approaches to prepare and characterise vacuoles from the filamentous fungus Ashbya gossypii are described, i.e. the isolation of vacuoles from hyphal cells and the controlled permeabilisation of the plasma membrane. By mechanical lysis of protoplasts and separation of the organelles on a stepped density gradient, we obtained a vacuolar fraction virtually free of contamination by other organelles, unlysed protoplasts and cell debris. The integrity of the isolated organelles was characterised by vital-staining, the presence of α-mannosidase, and retained accumulation of basic amino acids. In a second approach, the cell membrane of the fungus was selectively permeabilised by use of the saponin digitonin leaving the vacuoles in their physiological surrounding, i.e. protected by the rigid cell wall. The permeabilisation was monitored by the latency of predominantly cytosolic amino acids and the ATP status of the cells. Functional intactness of the vacuoles within the permeabilised hyphae was demonstrated by maintenance of the pH gradient across the vacuolar membrane as detected by accumulation of the fluorescent dye, Acridine orange. These two methods are well-suited tools for the in situ assay of intracellular compartmentation of metabolites, for vacuolar transmembrane fluxes in Ashbya gossypii, as well as for the direct access to vacuolar membranes and enzymes of this fungus.

Cell biologists sort things out: Analysis and purification of intracellular organelles by flow cytometry

Flow cytometry was established originally for measuring DNA content and for the analysis of cell-surface markers in combination with cell sorting. During the past two decades, it has added new dimensions to various areas of immunology and medicine. Increased sensitivity and precision of flow cytometers, accompanied by the development of new fluorescent dyes and probes, has led to new applications in molecular cell biology and genetics. This article focuses on applications of flow cytometry in analysis and sorting of intracellular organelles.

Purification of Organelles from Mammalian Cells

AbstractThe protocols in this unit illustrate a range of different procedures that have been used to fractionate tissue homogenates. They emphasize different fractionation techniques that have been used for rat liver, an abundant tissue that has been a favorite of many investigators and has served as the source of many organelle preparations of excellent purity. For selected procedures, other examples have been given using other tissue sources (e.g., glandular tissues that maintain protein storage granules for regulated secretion) or, where particularly favorable, cultured cells. The basis (or goal) of each separation and the merits and limitations of each procedure are summarized to provide a guide for selecting among the various approaches. The large number of protocols include specific details relevant to a particular sample cell type (and in many cases a particular organelle).

Subcellular distribution of multiple forms of glutathione reductase in leaves of pea (Pisum sativum L.)

On sodium-dodecyl-sulfate polyacrylamide gels, purified glutathione reductase (GR; EC 1.6.4.2) from the leaves of two- to three-week-old pea (Pisum sativum L. cv. Birte) seedlings was represented by a single band with an apparent molecular weight of 55 kilodaltons. This polypeptide was resolved to multiple isoforms by two-dimensional electrophoresis. Fractionation of protoplasts and purification of subcellular organelles has shown that enzyme activity is associated with the chloroplasts, mitochondria and cytosol (in this order, approx. 77%, 3%, and 20% of the total activity). Distinct multiple isoforms of the enzyme, which differed in isoelectric point and were compartment-specific, were resolved from purified mitochondria and chloroplasts. The latency of the glutathione reductase activity which co-purified on Percoll gradients with the mitochondrial marker enzyme, cytochrome-c oxidase (EC 1.9.3.1.), indicated that this enzyme was within the mitochondrion. The mitochondrial glutathione reductase activity was strongly dependent on NADPH and not NADH.

A rapid method for purification of organelles for DNA isolation: self-generated percoll gradients

Self-generated Percoll gradients have been used for rapid purification of crude chloroplasts and mitochondria, obtained by common differential centrifugation techniques. Such purified organelles were used for isolating DNA from safflower (Carthamus tinctorius L.), carrot (Daucus carota L.), various Solanaceae, and numerous somatic hybrids. The method is simple, has the advantage of not requiring DNase, and is particularly well suited when only limited amounts of aseptically grown shoots are available. As judged by restriction enzyme analyses and chloroplast DNA cloning experiments, the DNAs are of sufficient purity for many molecular biological applications without CsCl gradient purification.

Partition of cell particles and macromolecules: Separation and purification of biomolecules, cell organelles, membranes and cells in aqueous polymer two phase systems and their use in biochemical analysis and biotechnology. P‐A. Albertsson. Third Edition, 1986, John Wiley and Sons, Chichester, £61.35 pages 346.

Partition of cell particles and macromolecules: Separation and purification of biomolecules, cell organelles, membranes and cells in aqueous polymer two phase systems and their use in biochemical analysis and biotechnology. P‐A. Albertsson. Third Edition, 1986, John Wiley and Sons, Chichester, £61.35 pages 346.

Partition of Cell Particles and Macromolecules. Separation and Purification of Biomolecules, Cell Organelles, Membranes, and Cells in Aqueous Polymer Two-Phase Systems and Their Use in Biochemical Analysis and Biotechnology.Per Ake Albertsson

<i>Partition of Cell Particles and Macromolecules. Separation and Purification of Biomolecules, Cell Organelles, Membranes, and Cells in Aqueous Polymer Two-Phase Systems and Their Use in Biochemical Analysis and Biotechnology.</i>Per Ake Albertsson