scholarly journals A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

10.3791/2208 ◽  
2010 ◽  
Author(s):  
Shoko Ueki ◽  
Benjamin L. Meyers ◽  
Farzana Yasmin ◽  
Vitaly Citovsky
Keyword(s):  
In Planta ◽  
Biolistic Bombardment ◽  
Macromolecular Transport ◽  
Transport Assay ◽  
A Cell

scholarly journals PIN FORMED 2 facilitates the transport of Arsenite in Arabidopsis thaliana

2019 ◽  
Author(s):  
Mohammad Arif Ashraf ◽  
Kana Umetsu ◽  
Olena Ponomarenko ◽  
Michiko Saito ◽  
Mohammad Aslam ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Health Hazard ◽  
Root Tip ◽  
Root Growth Inhibition ◽  
Cellular Transport ◽  
In Planta ◽  
Molecular Physiology ◽  
X Ray ◽  
Transport Assay ◽  
Arsenical Species

AbstractArsenic contamination is a major environmental issue as it may lead to serious health hazard. Reduced trivalent form of inorganic arsenic, arsenite, is in general more toxic to plants compared with the fully oxidized pentavalent arsenate. The uptake of arsenite in plants has been shown to be mediated through a large subfamily of plant aquaglyceroporins, nodulin 26-like intrinsic proteins (NIPs). However, the efflux mechanisms, as well as the mechanism of arsenite-induced root growth inhibition, remain poorly understood. Using molecular physiology, synchrotron imaging, and root transport assay approaches, we show that the cellular transport of trivalent arsenicals in Arabidopsis thaliana is strongly modulated by PIN FORMED 2 (PIN2) auxin efflux transporter. Direct transport assay using radioactive arsenite, X-ray fluorescence imaging (XFI) coupled with X-ray absorption spectroscopy (XAS), and ICP-MS analysis revealed that pin2 plants accumulate higher concentrations of arsenite in root compared to wild-type. At the cellular level, arsenite specifically targets intracellular cycling of PIN2 and thereby alters the cellular auxin homeostasis. Consistently, loss of PIN2 results in aresenite hypersensitivity in root. XFI coupled with XAS further revealed that loss of PIN2 results in specific accumulation of arsenical species, but not the other metals like iron, zinc or calcium in the root tip. Collectively, these results demonstrate that PIN2 serves as a putative transporter of arsenical species in planta.

Molecular aspects of hormonal regulation

2011 ◽  
pp. 29-33
Author(s):  
Shern L. Chew
Keyword(s):  
Hormonal Regulation ◽  
Protein Biosynthesis ◽  
Screening Method ◽  
High Yield ◽  
Macromolecular Transport ◽  
A Cell ◽  
Molecular Aspects ◽  
Protein Tagging ◽  
Two Hybrid

The wide molecular effects of hormones have complicated the understanding of how hormones work on a cell. The old view was of a linear signalling pathway from the receptor to the nucleus, thereby stimulating gene transcription. This view is probably an oversimplification. Hormones can not only regulate most of the molecular machines of the cell, certainly the transcription machinery, but also others. These machines perform and coordinate functions such as RNA and protein biosynthesis, macromolecular transport, cell division or death, and intracellular signalling. Physiological studies have shown that hormonal regulation is specific, yet flexible, and has the ability to generate feedback loops. Advances in genetics, cellular, and molecular biology, and biochemistry have allowed much new, and sometimes confusing, data on the mechanisms underlying hormonal regulation. Many advances have been due to methods of identifying and verifying networks of interactions between proteins. One example is the yeast two-hybrid system, an in vivo genetic screening method for such interactions. Another example is the use of protein tagging (e.g. with histidine residues) which can allow rapid and high-yield protein purification for biochemical studies. This chapter will briefly review some of the mechanisms of hormonal regulation.

scholarly journals In vitro reconstitution of exocytosis from plasma membrane and isolated secretory vesicles.

1985 ◽  
Vol 101 (6) ◽  
pp. 2263-2273 ◽  
Author(s):  
J H Crabb ◽  
R C Jackson
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Secretory Vesicles ◽  
Cytoplasmic Face ◽  
In Vitro Reconstitution ◽  
Transport Assay ◽  
Complex Preparation ◽  
A Cell ◽  
Vectorial Transport

We describe the reconstitution of exocytotic function through recombination of purified cortical secretory vesicles (CVs) and plasma membrane from sea urchin eggs. CVs were dislodged from a cell surface complex preparation by gentle homogenization in an isotonic dissociation buffer, and purified by differential centrifugation. CV-free plasma membrane fragments were obtained by mechanically dislodging CVs from cortical lawn (CL) preparations with a jet of CL isolation buffer. This procedure produced a "plasma membrane lawn" preparation, consisting of plasma membrane fragments attached via their vitelline layer (an extracellular glycocalyx) to a polylysine-coated microscope slide. When freshly prepared CVs were incubated with plasma membrane lawns, CVs reassociated with the cytoplasmic face of the plasma membrane, forming an exocytotically competent, reconstituted cortical lawn (RL). Exocytosis in RLs was monitored by phase-contrast microscopy, and quantitated with a sensitive microphotometric assay. Half-maximal exocytosis in RLs occurred at 18.5 microM free Ca2+; half-maximal exocytosis in control lawns occurred at 5.7 microM free Ca2+. Greater than 90% of the purified CVs that were not attached to a plasma membrane lawn remained intact when bathed in a buffer containing millimolar Ca2+. This result excluded the possibility that Ca2+-triggered CV lysis was responsible for our observations, and confirmed that the association of CVs with the plasma membrane was required for exocytosis in RLs. Evidence that the Ca2+-stimulated release of CV contents in CLs and RLs is the in vitro equivalent of exocytosis was obtained with an immunofluorescence-based vectorial transport assay, using an antiserum directed against a CV content protein: stimulation of RLs or partially CV-depleted CLs with Ca2+ resulted in fusion of the CV and plasma membranes, and the vectorial transport of CV contents from the cytoplasmic to the extracytoplasmic face of the egg plasma membrane.

A cell-free approach to identify binding hotspots in plant immune receptors

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
George C. Markou ◽  
Casim A. Sarkar
Keyword(s):  
Ligand Binding ◽  
Hypersensitive Response ◽  
Polypeptide Chain ◽  
Trichoderma Viride ◽  
Leucine Rich Repeat ◽  
In Planta ◽  
Immune Receptors ◽  
A Cell ◽  
Modular Nature

AbstractPlant immune receptors are often difficult to express heterologously, hindering study of direct interactions between these receptors and their targets with traditional biochemical approaches. The cell-free method ribosome display (RD) enables expression of such recalcitrant proteins by keeping each nascent polypeptide chain tethered to its ribosome, which can enhance protein folding by virtue of its size and solubility. Moreover, in contrast to an in planta readout of receptor activity such as a hypersensitive response that conflates binding and signaling, RD enables direct probing of the interaction between plant immune receptors and their targets. Here, we demonstrate the utility of this approach using tomato recognition of Trichoderma viride ethylene-inducing xylanase (EIX) as a case study. Leveraging the modular nature of the tomato LeEIX2 and LeEIX1 leucine-rich repeat (LRR) receptors, we applied an entropy-informed algorithm to maximize the information content in our receptor segmentation RD experiments to identify segments implicated in EIX binding. Unexpectedly, two distinct EIX-binding hotspots were discovered on LeEIX2 and both hotspots are shared with decoy LeEIX1, suggesting that their contrasting receptor functions are not due to differential modes of ligand binding. Given that most plant immune receptors are thought to engage targets via their LRR sequences, this approach should be of broad utility in rapidly identifying their binding hotspots.

Characterization of a cell-surface antigen isolated from the plant pathogen Phytophthora parasitica var. nicotianae

1995 ◽  
Vol 73 (S1) ◽  
pp. 1104-1108 ◽  
Author(s):  
N. Séjalon ◽  
R. Dargent ◽  
F. Villalba ◽  
A. Bottin ◽  
M. Rickauer ◽  
...  
Keyword(s):  
Cell Wall ◽  
Synthetic Medium ◽  
Immunogold Labelling ◽  
Phytophthora Parasitica ◽  
In Planta ◽  
Black Shank ◽  
A Cell ◽  
Defence Responses

The genus Phytophthora contains several species that are pathogenic to plants. Phytophthora parasitica var. nicotianae is the causal agent of the black shank disease of tobacco. From this fungus we have isolated and purified to homogeneity a 34-kDa glycoprotein (GP34) that elicits defence responses in tobacco. Among other features, this glycoprotein contains the rare amino acid hydroxyproline. Antibodies against GP34 permitted us to study its localization in vitro and in planta. Ultrastructural cytochemistry using immunogold labelling shows that GP34 is present in the cell wall into which it is secreted by vesicles when the fungus is grown on synthetic medium. In zoospores, labelling precedes and is strictly associated with the formation of a new cell wall. At early stages of infection of tobacco, only a faint labelling of the mycelium is observed. Later on it is enhanced in the incompatible interaction between the fungus and a resistant host cultivar. Key words: cell wall, elicitor, hydroxyproline, Phytophthora, tobacco.

scholarly journals Watching Rafts Move Within Cells: A Fluorescence Microscope-Based Transport Assay

2000 ◽  
Vol 8 (1) ◽  
pp. 3-7
Author(s):  
Stephen W. Carmichael ◽  
Jeffery L. Salisbury
Keyword(s):  
Animal Model ◽  
Intact Animal ◽  
Retrograde Transport ◽  
Fluorescence Microscope ◽  
Anterograde Transport ◽  
Transport Assay ◽  
A Cell

Imagine a raft in a canal between point A and point B. On that raft is a visible (fluorescent) cargo. Also, attached to that raft is a motor that will propel the raft only from A to B (anterograde transport). When the raft gets to point B, another motor is attached that can propel the raft, and its cargo, and the anterograde motor, back to point A (retrograde transport). Within a cell, the canals are microtubLiles, and a lot is known about anterograde and retrograde transport in some systems, but these phenomena have not been directly observed in a living, intact animal. Until now, that is. In a pair of very interesting papers, the laboratory of Jonathan Scholey has shown us convincing micrographs of anterograde and retrograde transport in an important animal model.

scholarly journals GSK3 activity is a cell fate switch that balances the ratio of vascular cell type

2020 ◽  
Author(s):  
Takayuki Tamaki ◽  
Satoyo Oya ◽  
Makiko Naito ◽  
Yasuko Ozawa ◽  
Tomoyuki Furuya ◽  
...  
Keyword(s):  
Cell Fate ◽  
Culture System ◽  
Kinase Activity ◽  
Cell Type ◽  
In Planta ◽  
Vascular Cell ◽  
Signalling Molecules ◽  
Comparative Expression Analysis ◽  
Companion Cells ◽  
A Cell

AbstractThe phloem transports photosynthetic assimilates and signalling molecules. It mainly consists of sieve elements (SEs), which act as “highways” for transport, and companion cells (CCs), which serve as “gates” to load/unload cargos. Though SEs and CCs function together, it remains unknown what determines the ratio of SE/CC in the phloem. In this study, we develop a novel culture system for CC differentiation named VISUAL-CC, which reconstitutes the SE-CC complex formation. Comparative expression analysis in VISUAL-CC reveals that SE and CC differentiation tends to show negative correlation, while total phloem differentiation is unchanged. This varying SE/CC ratio is largely dependent on GSK3 kinase activity. Indeed, gsk3 hextuple mutants possess much more SEs and less CCs in planta. Conversely, gsk3 gain-of-function mutants induced by phloem-specific promoter partially increased the CC ratio. Taken together, GSK3 activity appears to function as a cell fate switch in the phloem, thereby balancing the SE/CC ratio.

Effect of Niacin on Mitochondria of Allium Cepa Root Cells

1967 ◽  
Vol 25 ◽  
pp. 78-79
Author(s):  
M. Arif Hayat
Keyword(s):  
Nicotinamide Adenine Dinucleotide ◽  
Hydrogen Transfer ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Nicotinamide Adenine ◽  
Root Tips ◽  
Root Cells ◽  
Transfer Processes ◽  
Standard Procedures ◽  
A Cell ◽  
Critical Interest

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.

Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

1978 ◽  
Vol 36 (2) ◽  
pp. 650-651
Author(s):  
Raul I. Garcia ◽  
Evelyn A. Flynn ◽  
George Szabo
Keyword(s):  
Direct Injection ◽  
Skin Pigmentation ◽  
Freeze Fracture ◽  
Pigment Granule ◽  
Time Lapse ◽  
Ultrastructural Level ◽  
Lanthanum Tracer ◽  
A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

Dendritic cells of the human epidermis: immuno-electron microscopic studies of la antigen reactivity

1978 ◽  
Vol 36 (2) ◽  
pp. 644-645
Author(s):  
G. Rowden ◽  
M. G. Lewis ◽  
T. M. Phillips
Keyword(s):  
Dendritic Cells ◽  
Langerhans Cells ◽  
Cell Types ◽  
Cell Type ◽  
Electron Microscopic ◽  
La Antigen ◽  
Microscopic Studies ◽  
A Cell ◽  
C3 Receptors ◽  
Antigen Reactivity

Langerhans cells of mammalian stratified squamous epithelial have proven to be an enigma since their discovery in 1868. These dendritic suprabasal cells have been considered as related to melanocytes either as effete cells, or as post divisional products. Although grafting experiments seemed to demonstrate the independence of the cell types, much confusion still exists. The presence in the epidermis of a cell type with morphological features seemingly shared by melanocytes and Langerhans cells has been especially troublesome. This so called "indeterminate", or " -dendritic cell" lacks both Langerhans cells granules and melanosomes, yet it is clearly not a keratinocyte. Suggestions have been made that it is related to either Langerhans cells or melanocyte. Recent studies have unequivocally demonstrated that Langerhans cells are independent cells with immune function. They display Fc and C3 receptors on their surface as well as la (immune region associated) antigens.

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