Particle bombardment and subcellular protein localization analysis in the aquatic plant Egeria densa

10.7287/peerj.preprints.3164 ◽

2017 ◽

Author(s):

Yasuhide Osaki ◽

Yutaka Kodama

Keyword(s):

Subcellular Localization ◽

Particle Bombardment ◽

Aquatic Plant ◽

Higher Plants ◽

Cell Layer ◽

Protein Localization ◽

Epidermal Cells ◽

Egeria Densa ◽

Easy Method ◽

Localization Analysis

Particle bombardment is a powerful and relatively easy method for transient expression of genes of interest in plant cells, especially those that are recalcitrant to other transformation methods. This method has facilitated numerous analyses of subcellular localization of fluorescent fusion protein constructs. Particle bombardment delivers genes to the first layer of plant tissue. In leaves of higher plants, epidermal cells are the first cell layer. Many studies have used the epidermal cell layer of onion bulb (Allium cepa) as the experimental tissue, because these cells are relatively large. However, onion epidermal cells lack developed plastids (i.e., chloroplasts), thereby precluding subcellular localization analysis of chloroplastic proteins. In this study, we developed a protocol for particle bombardment of the aquatic plant Egeria densa, and showed that it is a useful system for subcellular localization analysis of higher plant proteins. E. densa leaflets contain only two cell layers, and cells in the adaxial layer are sufficiently large for observation. The cells in both layers contain well-developed chloroplasts. We fused fluorescent proteins to conventional plant localization signals for the nucleus, cytosol, mitochondria, peroxisome, and chloroplast, and used particle bombardment to transiently express these fusion constructs in E. densa leaves. The plant subcellular localization signals functioned normally and displayed the expected distributions in transiently transformed E. densa cells, and even chloroplastic structures could be clearly visualized.

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Particle bombardment and subcellular protein localization analysis in the aquatic plant Egeria densa

PeerJ ◽

10.7717/peerj.3779 ◽

2017 ◽

Vol 5 ◽

pp. e3779 ◽

Cited By ~ 5

Author(s):

Yasuhide Osaki ◽

Yutaka Kodama

Keyword(s):

Subcellular Localization ◽

Particle Bombardment ◽

Aquatic Plant ◽

Higher Plants ◽

Cell Layer ◽

Protein Localization ◽

Epidermal Cells ◽

Egeria Densa ◽

Easy Method ◽

Localization Analysis

Particle bombardment is a powerful and relatively easy method for transient expression of genes of interest in plant cells, especially those that are recalcitrant to other transformation methods. This method has facilitated numerous analyses of subcellular localization of fluorescent fusion protein constructs. Particle bombardment delivers genes to the first layer of plant tissue. In leaves of higher plants, epidermal cells are the first cell layer. Many studies have used the epidermal cell layer of onion bulb (Allium cepa) as the experimental tissue, because these cells are relatively large. However, onion epidermal cells lack developed plastids (i.e., chloroplasts), thereby precluding subcellular localization analysis of chloroplastic proteins. In this study, we developed a protocol for particle bombardment of the aquatic plant Egeria densa, and showed that it is a useful system for subcellular localization analysis of higher plant proteins. E. densa leaflets contain only two cell layers, and cells in the adaxial layer are sufficiently large for observation. The cells in both layers contain well-developed chloroplasts. We fused fluorescent proteins to conventional plant localization signals for the nucleus, cytosol, mitochondria, peroxisome, and chloroplast, and used particle bombardment to transiently express these fusion constructs in E. densa leaves. The plant subcellular localization signals functioned normally and displayed the expected distributions in transiently transformed E. densa cells, and even chloroplastic structures could be clearly visualized.

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Comparative Subcellular Localization Analysis of Magnetosome Proteins Reveals a Unique Localization Behavior of Mms6 Protein onto Magnetite Crystals

Journal of Bacteriology ◽

10.1128/jb.00280-16 ◽

2016 ◽

Vol 198 (20) ◽

pp. 2794-2802 ◽

Cited By ~ 9

Author(s):

Atsushi Arakaki ◽

Daiki Kikuchi ◽

Masayoshi Tanaka ◽

Ayana Yamagishi ◽

Takuto Yoda ◽

...

Keyword(s):

Subcellular Localization ◽

Fluorescent Protein ◽

Protein Localization ◽

Linear Structure ◽

Magnetotactic Bacteria ◽

Regulation Mechanism ◽

Crystal Formation ◽

Spatial Regulation ◽

Magnetite Crystal ◽

Localization Analysis

ABSTRACTThe magnetosome is an organelle specialized for inorganic magnetite crystal synthesis in magnetotactic bacteria. The complex mechanism of magnetosome formation is regulated by magnetosome proteins in a stepwise manner. Protein localization is a key step for magnetosome development; however, a global study of magnetosome protein localization remains to be conducted. Here, we comparatively analyzed the subcellular localization of a series of green fluorescent protein (GFP)-tagged magnetosome proteins. The protein localizations were categorized into 5 groups (short-length linear, middle-length linear, long-length linear, cell membrane, and intracellular dispersing), which were related to the protein functions. Mms6, which regulates magnetite crystal growth, localized along magnetosome chain structures under magnetite-forming (microaerobic) conditions but was dispersed in the cell under nonforming (aerobic) conditions. Correlative fluorescence and electron microscopy analyses revealed that Mms6 preferentially localized to magnetosomes enclosing magnetite crystals. We suggest that a highly organized spatial regulation mechanism controls magnetosome protein localization during magnetosome formation in magnetotactic bacteria.IMPORTANCEMagnetotactic bacteria synthesize magnetite (Fe3O4) nanocrystals in a prokaryotic organelle called the magnetosome. This organelle is formed using various magnetosome proteins in multiple steps, including vesicle formation, magnetosome alignment, and magnetite crystal formation, to provide compartmentalized nanospaces for the regulation of iron concentrations and redox conditions, enabling the synthesis of a morphologically controlled magnetite crystal. Thus, to rationalize the complex organelle development, the localization of magnetosome proteins is considered to be highly regulated; however, the mechanisms remain largely unknown. Here, we performed comparative localization analysis of magnetosome proteins that revealed the presence of a spatial regulation mechanism within the linear structure of magnetosomes. This discovery provides evidence of a highly regulated protein localization mechanism for this bacterial organelle development.

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An efficient transient assays system using Agrobacterium-mediated transformation of onion (Allium cepa) epidermal cells

Indian Journal of Genetics and Plant Breeding (The) ◽

10.31742/ijgpb.80.3.17 ◽

2020 ◽

Vol 80 (03) ◽

Author(s):

Yu-Miao Zhang ◽

Jun Wang ◽

Tao Wu

Keyword(s):

Subcellular Localization ◽

Protein Interaction ◽

Protein Interactions ◽

Epidermal Cells ◽

Cyclin Dependent Kinase ◽

Protein Subcellular Localization ◽

Protein Protein Interactions ◽

Efficient System ◽

Protein Protein Interaction ◽

Onion Epidermal Cells

In this study, the Agrobacterium infection medium, infection duration, detergent, and cell density were optimized. The sorghum-based infection medium (SbIM), 10-20 min infection time, addition of 0.01% Silwet L-77, and Agrobacterium optical density at 600 nm (OD600), improved the competence of onion epidermal cells to support Agrobacterium infection at >90% efficiency. Cyclin-dependent kinase D-2 (CDKD-2) and cytochrome c-type biogenesis protein (CYCH), protein-protein interactions were localized. The optimized procedure is a quick and efficient system for examining protein subcellular localization and protein-protein interaction.

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Influence of Subcellular Localization and Functional State on Protein Turnover

Cells ◽

10.3390/cells10071747 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1747

Author(s):

Roya Yousefi ◽

Kristina Jevdokimenko ◽

Verena Kluever ◽

David Pacheu-Grau ◽

Eugenio F. Fornasiero

Keyword(s):

Subcellular Localization ◽

Functional State ◽

Protein Turnover ◽

Protein Localization ◽

Small Gtpase ◽

Turnover Rates ◽

Cellular Behavior ◽

Two Factors ◽

Brain Creatine Kinase ◽

Selection Of

Protein homeostasis is an equilibrium of paramount importance that maintains cellular performance by preserving an efficient proteome. This equilibrium avoids the accumulation of potentially toxic proteins, which could lead to cellular stress and death. While the regulators of proteostasis are the machineries controlling protein production, folding and degradation, several other factors can influence this process. Here, we have considered two factors influencing protein turnover: the subcellular localization of a protein and its functional state. For this purpose, we used an imaging approach based on the pulse-labeling of 17 representative SNAP-tag constructs for measuring protein lifetimes. With this approach, we obtained precise measurements of protein turnover rates in several subcellular compartments. We also tested a selection of mutants modulating the function of three extensively studied proteins, the Ca2+ sensor calmodulin, the small GTPase Rab5a and the brain creatine kinase (CKB). Finally, we followed up on the increased lifetime observed for the constitutively active Rab5a (Q79L), and we found that its stabilization correlates with enlarged endosomes and increased interaction with membranes. Overall, our data reveal that both changes in protein localization and functional state are key modulators of protein turnover, and protein lifetime fluctuations can be considered to infer changes in cellular behavior.

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Non-cell-autonomous function of the Antirrhinum floral homeotic proteins DEFICIENS and GLOBOSA is exerted by their polar cell-to-cell trafficking

Development ◽

10.1242/dev.122.11.3433 ◽

1996 ◽

Vol 122 (11) ◽

pp. 3433-3441 ◽

Cited By ~ 8

Author(s):

M.C. Perbal ◽

G. Haughn ◽

H. Saedler ◽

Z. Schwarz-Sommer

Keyword(s):

Cell Layer ◽

Epidermal Cells ◽

Molecular Techniques ◽

Cell Trafficking ◽

Mads Box ◽

Organ Identity ◽

Cell Layers ◽

Periclinal Chimeras ◽

The Difference ◽

Cell Autonomy

In Antirrhinum majus, petal and stamen organ identity is controlled by two MADS-box transcription factors, DEFICIENS and GLOBOSA. Mutations in either of these genes result in the replacement of petals by sepaloid organs and stamens by carpelloid organs. Somatically stable def and glo periclinal chimeras, generated by transposon excision events, were used to study the non-cell-autonomous functions of these two MADS-box proteins. Two morphologically distinct types of chimeras were analysed using genetic, morphological and molecular techniques. Restoration of DEF expression in the L1 cell layer results in the reestablishment of DEF and GLO functions in L1-derived cells only; inner layer cells retain their mutant sepaloid features. Nevertheless, this activity is sufficient to allow the expansion of petal lobes, highlighting the role of DEF in the stimulation of cell proliferation and/or cell shape and elongation when expressed in the L1 layer. Establishment of DEF or GLO expression in L2 and L3 cell layers is accompanied by the recovery of petaloid identity of the epidermal cells but it is insufficient to allow petal lobe expansion. We show by in situ immunolocalisation that the non-cell-autonomy is due to direct trafficking of DEF and GLO proteins from the inner layer to the epidermal cells. At least for DEF, this movement appears to be polar since DEF acts cell-autonomously when expressed in the L1 cell layer. Furthermore, the petaloid revertant sectors observed on second whorl mutant organs and the mutant margins of petals of L2L3 chimeras suggest that DEF and GLO intradermal movement is limited. This restriction may reflect the difference in the regulation of primary plasmodesmata connecting cells from the same layer and secondary plasmodesmata connecting cells from different layers. We propose that control of intradermal trafficking of DEF and GLO could play a role in maintaining of the boundaries of their expression domains.

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Cutaneous DNA Vaccination Against Ebola Virus by Particle Bombardment: Histopathology and Alteration of CD3-positive Dendritic Epidermal Cells

Veterinary Pathology ◽

10.1354/vp.38-2-203 ◽

2001 ◽

Vol 38 (2) ◽

pp. 203-215 ◽

Cited By ~ 19

Author(s):

K. E. Steele ◽

K. Stabler ◽

L. VanderZanden

Keyword(s):

Particle Bombardment ◽

Guinea Pigs ◽

Ebola Virus ◽

Dna Vaccination ◽

Hair Follicles ◽

Epidermal Cells ◽

Gold Particles ◽

Immunocompetent Mice ◽

Inflammatory Changes ◽

Epidermal Necrosis

We analyzed the localization of gold particles, expression of immunogenic protein, and histopathologic changes after vaccinating guinea pigs and mice with a DNA vaccine to the Ebola virus glycoprotein administered by cutaneous particle bombardment. Gold particles were deposited in all layers of the epidermis and in the dermis. Those in the epidermis were lost as the damaged layers sloughed, while those in the dermis were phagocytized by macrophages. Glycoprotein was demonstrated by immunohistochemistry primarily in keratinocytes in the epidermis and hair follicle epithelium and less frequently in dermal macrophages, fibroblasts, sebocytes, and cells that appeared to be Langerhans cells. The number of cells that expressed glycoprotein increased between 4 and 8 hours postvaccination, then decreased to near zero by 48 hours. The vaccine sites were histologically divisible into three zones. The central portion, zone 1, contained the most gold particles in the dermis and epidermis and had extensive tissue damage, including full-thickness epidermal necrosis. Zone 2 contained fewer gold particles in the epidermis and dermis and had less extensive necrosis. The majority of cells in which glycoprotein was expressed were in zone 2. Zone 3 contained gold particles only in the epidermis and had necrosis of only a few scattered cells. Regeneration of the epidermis in damaged areas was evident at 24 hours postvaccination and was essentially complete by day 5 in the mice and day 10 in the guinea pigs. Inflammatory changes were characterized by hemorrhage, edema, and infiltrates of neutrophils initially and by infiltrates of lymphocytes and macrophages at later times. In zone 1, inflammation affected both the epidermis and dermis. Peripherally, inflammation was relatively limited to the epidermis. CD3-positive dendritic epidermal cells were demonstrated in the epidermis and superficial hair follicles of unvaccinated immunocompetent mice and beige mice but not of SCID mice. These cells disappeared from all but the most peripheral portions of the vaccine sites of vaccinated mice within 24 hours. They reappeared slowly, failing to reach numbers comparable with unvaccinated mice by 35 days postvaccination. The epidermis of control guinea pigs also had CD3-positive cells, but they did not have dendrites. These findings should contribute to a better understanding of the mechanisms operating in response to DNA vaccination by particle bombardment.

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