Discontinuous Sucrose Gradient Research Articles

Chloroplast Envelope Membrane Subfractionation from Arabidopsis and Pea.

Due to their endosymbiotic origin, chloroplasts harbor several subcompartments and membrane systems. Each of these has a different protein and lipid composition that dynamically changes either naturally during plant development or induced by environmental stimuli. Here, we describe a protocol for chloroplast envelope membrane subfractionation via discontinuous sucrose gradients, which offers the possibility to separate the different plastid subcompartments for several downstream applications. It is a strong tool for protein sublocalization studies as well as for tracking dynamic movement patterns. Furthermore, it can be combined with in vitro import studies of radioactively labeled proteins, which allows sublocalization of putative envelope proteins independent of the availability of specific antisera.

Combining BN-PAGE and microscopy techniques to investigate pigment-protein complexes and plastid transitions in citrus fruit

BackgroundChlorophyll and carotenoids, the most widely distributed lipophilic pigments in plants, contribute to fruit coloration during development and ripening. These pigments are assembled with pigment-protein complexes localized at plastid membrane. Pigment-protein complexes are essential for multiple cellular processes, however, their identity and composition in fruit have yet to be characterized.ResultsBy using BN-PAGE technique in combination with microscopy, we studied pigment-protein complexes and plastid transformation in the purified plastids from the exocarp of citrus fruit. The discontinuous sucrose gradient centrifugation was used to isolate total plastids from kumquat fruit, and the purity of isolated plastids was assessed by microscopy observation and western blot analysis. The isolated plastids at different coloring stages were subjected to pigment autofluorescence observation, western blot, two-dimensional electrophoresis analysis and BN-PAGE assessment. Our results demonstrated that (i) chloroplasts differentiate into chromoplasts during fruit coloring, and this differentiation is accompanied with a decrease in the chlorophyll/carotenoid ratio; (ii) BN-PAGE analysis reveals the profiles of macromolecular protein complexes among different types of plastids in citrus fruit; and (iii) the degradation rate of chlorophyll-protein complexes varies during the transition from chloroplasts to chromoplasts, with the stability generally following the order of LHCII > PS II core > LHC I > PS I core.ConclusionsOur optimized methods for both plastid separation and BN-PAGE assessment provide an opportunity for developing a better understanding of pigment-protein complexes and plastid transitions in plant fruit. These attempts also have the potential for expanding our knowledge on the sub-cellular level synchronism of protein changes and pigment metabolism during the transition from chloroplasts to chromoplasts.

Efficient Isolation and Purification of High-Quality Arabidopsis thaliana Trichomes.

Trichomes are fine outgrowths on the surface of aerial plant organs which play a role in protecting plants against water loss, UV radiation, and herbivore feeding. Throughout the years, trichomes have become a popular paradigm in biological research. For example, trichomes on rosette leaves of the reference plant Arabidopsis thaliana have been used as a model to investigate cell development, cell differentiation, and, more recently, cell wall biogenesis. State of the art -omics studies on specific cell types or tissues often require physical separation, enrichment, and purification. This, of course, also applies to leaf trichomes, and various methods have thus been proposed to separate trichomes and leaf tissue. Though most of these methods are indeed suitable for trichome isolation, they suffer in part from tedious operating procedures, low yield, poor sample purity, and reduced trichome integrity. We have thus revised a previously reported method for trichome isolation, and report here an efficient and scalable procedure for the isolation and gradient centrifugation-based purification of high-quality A. thaliana trichomes. We describe the preparation of plant material and trichome release, which is based on prolonged gentle agitation of plant seedlings in the presence of a cation-chelating agent that weakens trichome-leaf interactions. We also outline the steps for the subsequent recovery and purification of the isolated crude trichome fraction, which is based on the use of discontinuous sucrose gradient centrifugation. In addition to A. thaliana, we have found that this procedure can be applied to release and enrich glandular and non-glandular trichomes from various species, including Solanum lycopersicum and Nicotiana benthamiana. The resulting purified leaf trichomes can be subjected to different types of bioassays, including histochemistry, biochemical quantification of cell wall monosaccharides, and transcriptomics, as well as proteomic profiling. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of plant material for release and enrichment of A. thaliana trichomes Basic Protocol 2: Purification of A. thaliana trichomes by density gradient centrifugation.

O-GlcNAcylation Inhibits Endocytosis of Amyloid Precursor Protein by Decreasing Its Localization in Lipid Raft Microdomains.

Like protein phosphorylation, O-GlcNAcylation is a common post-translational protein modification. We already reported that O-GlcNAcylation of amyloid precursor protein (APP) in response to insulin signaling reduces neurotoxic amyloid-β (Aβ) production via inhibition of APP endocytosis. Internalized APP is delivered to endosomes and lysosomes where Aβ is produced. However, the molecular mechanism involved in the effect of APP O-GlcNAcylation on APP trafficking remains unknown. To investigate the relationship between APP O-GlcNAcylation and APP endocytosis, we tested the effects of insulin on neuroblastoma SH-SY5Y cells overexpressing APP and BACE1, and cultured rat hippocampal neurons. The present study showed that APP O-GlcNAcylation translocated APP from lipid raft to non-raft microdomains in the plasma membrane by using immunocytochemistry and discontinuous sucrose gradients method. By using the biotinylation method, we also found that APP preferentially underwent endocytosis from lipid rafts and that the amount of internalized APP from lipid rafts was specifically reduced by O-GlcNAcylation. These results indicate that O-GlcNAcylation can regulate lipid raft-dependent APP endocytosis via translocation of APP into non-raft microdomains. Our findings showed a new functional role of O-GlcNAcylation for the regulation of APP trafficking, offering new mechanistic insight for Aβ production.

Rhodopsins build up the birefringent bodies of the dinoflagellate Oxyrrhis marina

The ultrastructure of the birefringent bodies of the dinoflagellate Oxyrrhis marina was investigated by transmission electron microscopy. Ultrathin sectioning revealed that the bodies consist of highly ordered and densely packed lamellae, which show a regular striation along their longitudinal axis. A lattice distance of 6.1 nm was measured for the densely packed lamellae by FFT (Fast Fourier Transformation) analysis. In addition, a rather faint and oblique running striation was registered. Lamellae sectioned rather oblique or almost close to the surface show a honeycombed structure with a periodicity of 7.2–7.8 nm. Freeze-fracture transmission electron microscopy revealed that the lamellae are composed of highly ordered, crystalline arrays of particles. Here, FFT analysis resulted in lattice distances of 7.0–7.6 nm. Freeze-fracture transmission electron microscopy further revealed that the bodies remained intact after cell rupture followed by ascending flotation of the membrane fractions on discontinuous sucrose gradients. The birefringent bodies most likely are formed by evaginations of membranes, which separate the cytoplasm from the food vacuoles. Distinct, slightly reddish-colored areas, which resembled the birefringent bodies with respect to size and morphology, were registered by bright field light microscopy within Oxyrrhis marina cells. An absorbance maximum at 540 nm was registered for these areas, indicating that they are composed of rhodopsins. This was finally proven by immuno-transmission electron microscopy, as antisera directed against the C-terminal amino acid sequences of the rhodopsins AEA49880 and ADY17806 intensely immunolabeled the birefringent bodies of Oxyrrhis marina.

Enrichment of microsomes from Chinese hamster ovary cells by subcellular fractionation for its use in proteomic analysis.

Chinese hamster ovary cells have been the workhorse for the production of recombinant proteins in mammalian cells. Since biochemical, cellular and omics studies are usually affected by the lack of suitable fractionation procedures to isolate compartments from these cells, differential and isopycnic centrifugation based techniques were characterized and developed specially for them. Enriched fractions in intact nuclei, mitochondria, peroxisomes, cis-Golgi, trans-Golgi and endoplasmic reticulum (ER) were obtained in differential centrifugation steps and subsequently separated in discontinuous sucrose gradients. Nuclei, mitochondria, cis-Golgi, peroxisomes and smooth ER fractions were obtained as defined bands in 30–60% gradients. Despite the low percentage represented by the microsomes of the total cell homogenate (1.7%), their separation in a novel sucrose gradient (10–60%) showed enough resolution and efficiency to quantitatively separate their components into enriched fractions in trans-Golgi, cis-Golgi and ER. The identity of these organelles belonging to the classical secretion pathway that came from 10–60% gradients was confirmed by proteomics. Data are available via ProteomeXchange with identifier PXD019778. Components from ER and plasma membrane were the most frequent contaminants in almost all obtained fractions. The improved sucrose gradient for microsomal samples proved being successful in obtaining enriched fractions of low abundance organelles, such as Golgi apparatus and ER components, for biochemical and molecular studies, and suitable for proteomic research, which makes it a useful tool for future studies of this and other mammalian cell lines.

Altered recruitment of Lyn, Syk and ZAP-70 into lipid rafts of activated B cells in Systemic Lupus Erythematosus

There is evidence that B cells from patients with Systemic Lupus Erythematosus (SLE) could be hyperactivated due to changes in their lipid rafts (LR) composition, leading to altered BCR-dependent signals. This study aimed to characterize possible alterations in the recruitment of protein tyrosine kinases (PTK) into B cells LR from SLE patients. Fifteen patients with SLE and ten healthy controls were included. Circulating B cells were isolated by negative selection and stimulated with goat Fab´2 anti-human IgM/IgG. LR were isolated with a non-ionic detergent and ultracentrifuged on 5–45% discontinuous sucrose gradients. Proteins from each fraction were analyzed by Western Blot. Total levels of Lyn, Syk, and ZAP-70 in resting B cells were similar in SLE patients and healthy controls. Upon BCR activation, Lyn, Syk and ZAP-70 recruitment into LR increased significantly in B cells of healthy controls and patients with inactive SLE. In contrast, in active SLE patients there was a great heterogeneity in the recruitment of signaling molecules and the recruitment of ZAP-70 was mainly observed in patients with decreased Syk recruitment into LR of activated B cells. The reduction in Flotilin-1 and Lyn recruitment in SLE patients seem to be associated with disease activity. These findings suggest that in SLE patients the PTK recruitment into B cell LR is dysregulated and that B cells are under constant activation through BCR signaling. The decrease of Lyn and Syk, the expression of ZAP-70 by B cells and the increase in Calcium fluxes in response to BCR stimulation in active SLE patients, further support that B cells from SLE patients are under constant activation through BCR signaling, as has been proposed.

Separation of cells in density gradients with temporary solidification of the gradient: Application to Escherichia coli forms obtained by microwave treatment of pure cultures

Gelatin added in certain concentration into the density gradient mixtures allows to solidify and melt the gradient reversibly. Such a method was applied to separation of partially disintegrated Escherichia coli VKM B-125 cells in discontinuous sucrose gradient which was optimized to obtain small round “nano-cells” free from other cell components.Gelatin added to the sucrose solutions had the advantages: i) accurate gradient layers were formed, ii) gelatin did not interfere with the cells and sub-particles during centrifugation, iii) solidified gelatin allowed to cut out the separated cell components, and iv) gelatin allowed to prepare ready-made solidified gradient centrifugation tubes into storage.

Human Follicle Stimulating Hormone Receptor Lipid Raft Residency is Hormone and Caveolin Dependent

Human follicle stimulating hormone (hFSH) is a gonadotropin responsible for the maturation of ovarian follicles in women and production of sperm in men. The receptor (hFSHR) is a G protein‐coupled receptor (GPCR); a class of receptors found embedded in the cell membrane to receive the hormone resulting in initiation of a complex downstream signaling cascade. Through previous research in the Cohen lab, hFSHR has been shown to be located within compartments of the cell membrane known as lipid rafts. Lipid rafts are microdomains of the membrane containing a higher concentration of cholesterol, glycosphingolipids, and a protein known as caveolin. Current research is focused on evaluating the mechanisms and effects of hFSHR lipid raft residency. Using discontinuous sucrose gradients we have shown that hFSH stimulation of an HEK293 cell line stably transfected with hFSHR cDNA results in alteration of hFSHR lipid raft residency in a time dependent manner.To better understand the mechanism and effect of hFSHR lipid raft residency the same cells were treated with a peptide mimetic of transmembrane domain 4 of hFSHR. This peptide includes a sequence consistent with a motif shown in other proteins to interact with caveolin (ϕXϕXXXXϕXXϕ where ϕ is an aromatic amino acid). The putative caveolin interaction motif of hFSHR was synthesized along with the HIV‐TAT leader sequence to allow passage through the membrane. A second peptide where the aromatic amino acids were replaced with leucine was also used. Cells treated with the wild type peptide showed elevated basal signaling in the absence of hFSH, an effect not observed with the mutant peptide.Together, these results suggest that hFSHR residency in lipid rafts is dependent on hormone treatment and interaction with caveolin. Through studying hFSHR lipid raft residency and signaling we hope to identify ways to control the receptor for improved methods for contraception and treatment of infertility.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A high-sensitivity photonic crystal fiber (PCF) based on the surface plasmon resonance (SPR) biosensor for detection of density alteration in non-physiological cells (DANCE)

• A novel highly sensitive photonic crystal fiber based on the surface plasmon resonance (PCF-SPR) biosensor for the detection of the density alteration in non-physiological cells (DANCE) was investigated. • Coating sensor with gold layer on the wall of the liquid channel not only overcame manufacturing challenges for experiments, but also enhanced the sensitivity. • According to simulation results, the biosensor shows a maximum amplitude sensitivity of 2000 nm/RIU and resolution as high as 5 × 10 −5 RIU. According to the wavelength interrogation method, a maximum spectral sensitivity of 9000 nm/RIU in the sensing range between 1.33 and 1.53 is achieved, corresponding to a resolution as high as 1.11 × 10 −5 RIU for the biosensor. A highly sensitive photonic crystal fiber based on the surface plasmon resonance (PCF-SPR) biosensor for the detection of the density alteration in non-physiological cells (DANCE) is described. Human acute leukemia cells are determined by the discontinuous sucrose gradient centrifugation (DSGC) in which the cells are separated into several bands. The separated cells with different intracellular densities and refractive indexes (RI) ranging from 1.3342 to 1.3344 are distinguished in situ by means of the differential transmission spectrum. The biosensor shows a maximum amplitude sensitivity of 2000 nm/RIU and resolution as high as 5 × 10 −5 RIU. According to the wavelength interrogation method, a maximum spectral sensitivity of 9000 nm/RIU in the sensing range between 1.33 and 1.53 is achieved, corresponding to a resolution as high as 1.11 × 10 −5 RIU for the biosensor. The proposed PCF-SPR biosensor has promising application in biological and biochemical detection.

The plasma membrane proteome of Medicago truncatula roots as modified by arbuscular mycorrhizal symbiosis.

In arbuscular mycorrhizal (AM) roots, the plasma membrane (PM) of the host plant is involved in all developmental stages of the symbiotic interaction, from initial recognition to intracellular accommodation of intra-radical hyphae and arbuscules. Although the role of the PM as the agent for cellular morphogenesis and nutrient exchange is especially accentuated in endosymbiosis, very little is known regarding the PM protein composition of mycorrhizal roots. To obtain a global overview at the proteome level of the host PM proteins as modified by symbiosis, we performed a comparative protein profiling of PM fractions from Medicago truncatula roots either inoculated or not with the AM fungus Rhizophagus irregularis. PM proteins were isolated from root microsomes using an optimized discontinuous sucrose gradient; their subsequent analysis by liquid chromatography followed by mass spectrometry (MS) identified 674 proteins. Cross-species sequence homology searches combined with MS-based quantification clearly confirmed enrichment in PM-associated proteins and depletion of major microsomal contaminants. Changes in protein amounts between the PM proteomes of mycorrhizal and non-mycorrhizal roots were monitored further by spectral counting. This workflow identified a set of 82 mycorrhiza-responsive proteins that provided insights into the plant PM response to mycorrhizal symbiosis. Among them, the association of one third of the mycorrhiza-responsive proteins with detergent-resistant membranes pointed at partitioning to PM microdomains. The PM-associated proteins responsive to mycorrhization also supported host plant control of sugar uptake to limit fungal colonization, and lipid turnover events in the PM fraction of symbiotic roots. Because of the depletion upon symbiosis of proteins mediating the replacement of phospholipids by phosphorus-free lipids in the plasmalemma, we propose a role of phosphate nutrition in the PM composition of mycorrhizal roots.

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization.

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.

Development of standard methods for Zika virus propagation, titration, and purification

The emergence of Zika virus (ZIKV) infection has stimulated several research groups to study and collaborate to understand virus biology and pathogenesis. These efforts may assist with the development of antiviral drugs, vaccines and diagnostic tests, as well as to promote advancements in public health policies. Here, we aim to develop standard protocols for propagation, titration, and purification of ZIKV strains, by systematically testing different cell types, kinetics, multiplicity of infection and centrifugation protocols. ZIKV produces a productive infection in human, non-human primate, and rodents-derived cell lines, with different efficacies. The highest yield of ZIKV-AFR and ZIKV-BR infectious progeny was obtained at 7days post infection in C6/36 cells (7×107 and 2×108 PFU/ml, respectively). However, high titers of ZIKV-AFR could be obtained at earlier time points in Vero cells (2.5×107PFU/ml at 72hpi), whereas ZIKV-BR titers reached 108 PFU/ml at 4dpi in C6/36 cells. High yield of purified virus was obtained by purification through a discontinuous sucrose gradient. This optimized procedure will certainly contribute to future studies of virus structure and vaccine development. Beyond the achievement of efficient virus propagation, the normalization of these protocols will also allow different laboratories around the world to better compare and discuss data regarding different features of ZIKV biology and disease, contributing to more efficient collaborations and progression in ZIKV research.

Discovery Recovery Methods and Detection of food borne parasites

In 2013, more than 640 individuals acquired Cyclospora infection in the US. Illness was associated with consumption of imported salad mixes and cilantro that were commercially processed and rinsed. To date, there is no protocol that would allow testing of large volumes of water to detect small number of parasites in produce wash‐waters. Cryptosporidium parvum and Cyclospora cayetanensis are protozoan parasites causing gastrointestinal illness in humans and animals both immunocompetent and immunocompromised hosts. Cryptosporidium parvum are spherical, 4–5μm in size, it can be acquired when ingesting foods and drinking, or accidentally ingesting, water contaminated with feces containing Cryptosporidium oocysts. The illnesses are characterized by acute diarrhea, stomach cramps, nausea, vomiting, and weight loss. Cyclospora cayetanensis are spherical as well, it is 8–10μm in size, it can also be acquired by person‐to‐person transmission and when in contact with infected animals; it is also spread via the fecal‐oral route (the host must ingest something that had been contaminated with feces that contain infectious oocysts). For oocysts to be termed infectious, the oocyst has to be sporulated (produce or form spores). The related disease Cyclosporiasis is acquired by ingestion of water or foods contaminated with human feces containing Cyclospora oocysts. Water samples were collected from a wash site in the southern area of Georgia every other day. The objective of this study was to compare two methods for oocyst recovery and detection from carrot wash‐water. The carrots were grated and washed with the water sample. The carrot wash‐water was spiked with ten and fifty oocysts of Cryptosporidium and Cyclospora. Two types of produce wash solutions fresh water and chlorinated water used by the fresh produce industry were evaluated ten liters of wash water were flocculated or filtered and then concentrated by centrifuging. Cryptosporidium oocysts from the concentrated water sample was treated with immunomagnetic separation (IMS) and detected by immunofluorescence assay (IFA). Cyclospora oocysts were concentrated by discontinuous sucrose gradients and examined by Nomarski microscopy. All samples were further examined by nested Polymerase Chain Reaction (PCR) specific for each parasite. Though both methods effectively recovered parasites, our trial also suggests that filtration and flocculation does not completely remove Cryptosporidium and Cyclospora oocysts in the wash water. In conclusion, these methods can significantly contribute to environmental testing during outbreak investigations and surveillance studies.Support or Funding InformationNSF #1067896

Characterization of lipid rafts in human platelets using nuclear magnetic resonance: A pilot study

Lipid microdomains (‘lipid rafts’) are plasma membrane subregions, enriched in cholesterol and glycosphingolipids, which participate dynamically in cell signaling and molecular trafficking operations. One strategy for the study of the physicochemical properties of lipid rafts in model membrane systems has been the use of nuclear magnetic resonance (NMR), but until now this spectroscopic method has not been considered a clinically relevant tool. We performed a proof-of-concept study to test the feasibility of using NMR to study lipid rafts in human tissues. Platelets were selected as a cost-effective and minimally invasive model system in which lipid rafts have previously been studied using other approaches. Platelets were isolated from plasma of medication-free adult research participants (n=13) and lysed with homogenization and sonication. Lipid-enriched fractions were obtained using a discontinuous sucrose gradient. Association of lipid fractions with GM1 ganglioside was tested using HRP-conjugated cholera toxin B subunit dot blot assays. 1H high resolution magic-angle spinning nuclear magnetic resonance (HRMAS NMR) spectra obtained with single-pulse Bloch decay experiments yielded spectral linewidths and intensities as a function of temperature. Rates of lipid lateral diffusion that reported on raft size were measured with a two-dimensional stimulated echo longitudinal encode-decode NMR experiment. We found that lipid fractions at 10–35% sucrose density associated with GM1 ganglioside, a marker for lipid rafts. NMR spectra of the membrane phospholipids featured a prominent ‘centerband’ peak associated with the hydrocarbon chain methylene resonance at 1.3ppm; the linewidth (full width at half-maximum intensity) of this ‘centerband’ peak, together with the ratio of intensities between the centerband and ‘spinning sideband’ peaks, agreed well with values reported previously for lipid rafts in model membranes. Decreasing temperature produced decreases in the 1.3ppm peak intensity and a discontinuity at ~18°C, for which the simplest explanation is a phase transition from Ld to Lo phases indicative of raft formation. Rates of lateral diffusion of the acyl chain lipid signal at 1.3ppm, a quantitative measure of microdomain size, were consistent with lipid molecules organized in rafts. These results show that HRMAS NMR can characterize lipid microdomains in human platelets, a methodological advance that could be extended to other tissues in which membrane biochemistry may have physiological and pathophysiological relevance.

Isolation of Lipid Rafts Through Discontinuous Sucrose Gradient Centrifugation and Fas/CD95 Death Receptor Localization in Raft Fractions.

Lipid raft domains, enriched in sphingolipids and cholesterol, serve as sorting platforms and hubs for signal transduction proteins, and show resistance to detergent solubilization. Despite rafts have been involved in survival processes, these membrane domains have also been shown to play a major role in the modulation of death receptor signaling. Here, we describe a detailed protocol for isolating lipid rafts from whole cells by taking advantage of the lipid raft resistance to Triton X-100 solubilization at 4 °C, followed by sucrose gradient centrifugation, with subsequent analysis of Fas/CD95 death receptor localization in the raft fractions by immunoblotting. This method is also useful to localize additional proteins in membrane rafts.

Biochemical Isolation of Myonuclei from Mouse Skeletal Muscle Tissue.

Skeletal muscle provides the contractile force necessary for movement, swallowing, and breathing and, consequently, is necessary for survival. Skeletal muscle cells are unique in that they are extremely large cells containing thousands of nuclei. These nuclei must all work in concert to maintain skeletal muscle function and thereby maintain life. The nucleus is a major site of signaling integration and gene expression regulation. However, examining nuclear processes in skeletal muscle can be difficult because myonuclei are challenging to isolate. We optimized a protocol to purify myonuclei from whole muscle tissue using ultracentrifugation over a discontinuous sucrose gradient to separate the nuclear fraction. We used these purified nuclei for downstream applications including flow cytometry and mass spectrometry. We used this method to compare the myonuclear proteome of young and old mouse hindlimb muscles (Cutler et al., 2017). This protocol may be applied to isolating myonuclei for a variety of downstream analyses such as flow cytometry, microscopy, Western blot, and proteomics.

Comparative "Golgi" Proteome Study of Lolium multiflorum and Populus trichocarpa.

The Golgi apparatus (GA) is a crucial organelle in the biosynthesis of non-cellulosic polysaccharides, glycoproteins and proteoglycans that are primarily destined for secretion to the cell surface (plasma membrane, cell wall and apoplast). Only a small proportion of the proteins involved in these processes have been identified in plants, with the majority of their functions still unknown. The availability of a GA proteome would greatly assist plant biochemists, cell and molecular biologists in determining the precise function of the cell wall-related proteins. There has been some progress towards defining the GA proteome in the model plant system Arabidopsis thaliana, yet in commercially important species, such as either the cereals or woody species there has been relatively less progress. In this study, we applied discontinuous sucrose gradient centrifugation to partially enrich GA from suspension cell cultures (SCCs) and combined this with stable isotope labelling (iTRAQ) to determine protein sub-cellular locations. Results from a representative grass species, Italian ryegrass (Lolium multiflorum) and a dicot species, black cottonwood (Populus trichocarpa) are compared. The results confirm that membrane fractionation approaches that provide effective GA-enriched fractions for proteomic analyses in Arabidopsis are much less effective in the species examined here and highlight the complexity of the GA, both within and between species.

Protein composition of 6K2-induced membrane structures formed during Potato virus A infection.

The definition of the precise molecular composition of membranous replication compartments is a key to understanding the mechanisms of virus multiplication. Here, we set out to investigate the protein composition of the potyviral replication complexes. We purified the potyviral 6K2 protein-induced membranous structures from Potato virus A (PVA)-infected Nicotiana benthamiana plants. For this purpose, the 6K2 protein, which is the main inducer of potyviral membrane rearrangements, was expressed in fusion with an N-terminal Twin-Strep-tag and Cerulean fluorescent protein (SC6K) from the infectious PVA cDNA. A non-tagged Cerulean-6K2 (C6K) virus and the SC6K protein alone in the absence of infection were used as controls. A purification scheme exploiting discontinuous sucrose gradient centrifugation followed by Strep-tag-based affinity chromatography was developed. Both (+)- and (-)-strand PVA RNA and viral protein VPg were co-purified specifically with the affinity tagged PVA-SC6K. The purified samples, which contained individual vesicles and membrane clusters, were subjected to mass spectrometry analysis. Data analysis revealed that many of the detected viral and host proteins were either significantly enriched or fully specifically present in PVA-SC6K samples when compared with the controls. Eight of eleven potyviral proteins were identified with high confidence from the purified membrane structures formed during PVA infection. Ribosomal proteins were identified from the 6K2-induced membranes only in the presence of a replicating virus, reinforcing the tight coupling between replication and translation. A substantial number of proteins associating with chloroplasts and several host proteins previously linked with potyvirus replication complexes were co-purified with PVA-derived SC6K, supporting the conclusion that the host proteins identified in this study may have relevance in PVA replication.

Lipid Extraction from HeLa Cells, Quantification of Lipids, Formation of Large Unilamellar Vesicles (LUVs) by Extrusion and in vitro Protein-lipid Binding Assays, Analysis of the Incubation Product by Transmission Electron Microscopy (TEM) and by Flotation across a Discontinuous Sucrose Gradient

Lipid Extraction from HeLa Cells, Quantification of Lipids, Formation of Large Unilamellar Vesicles (LUVs) by Extrusion and in vitro Protein-lipid Binding Assays, Analysis of the Incubation Product by Transmission Electron Microscopy (TEM) and by Flotation across a Discontinuous Sucrose Gradient