Ultrastructural distribution of a MAP kinase and transcripts in quiescent and cycling plant cells and pollen grains

1999 ◽  
Vol 112 (7) ◽  
pp. 1065-1076
Author(s):  
G. Prestamo ◽  
P.S. Testillano ◽  
O. Vicente ◽  
P. Gonzalez-Melendi ◽  
M.J. Coronado ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Polyclonal Antibodies ◽  
Plant Cells ◽  
Pollen Grains ◽  
Cell Types ◽  
Protein Distribution ◽  
Animal Cells ◽  
Proliferating Cells ◽  
Quiescent Cells

Mitogen-activated protein kinases (MAPKs) are components of a kinase module that plays a central role in the transduction of diverse extracellular stimuli, including mitogens, specific differentiation and developmental signals and stress treatments. This shows that reversible protein phosphorylation cascades play a pivotal role in signal transduction in animal cells and yeast, particularly the entry into mitosis of arrested cells. Homologues of MAPKs have been found and cloned in various plant species, but there have been no data about their in situ localization at the subcellular level and their expression in plant cells so far. In the present paper we report the first data on the ultrastructural in situ localization of MAPK and their mRNAs in various plant cells. Proliferating and quiescent meristematic plant cells were studied to evaluate whether changes in MAPK presence, distribution and expression accompany the entry into proliferation of dormant cells. Moreover, MAPK localization was analyzed in vacuolate microspores. Polyclonal antibodies against the deduced MAPK from the tobacco Ntf6 clone were able to recognize homologue epitopes by immunocytochemical techniques in the cell types studied. The pattern of protein distribution is similar in all the cases studied: it is localized in the cytoplasm and in the nucleus, mainly in the interchromatin region. The quantitative study of the density showed that MAPK labelling is more abundant in cycling than in quiescent cells, also suggesting that, in plants, MAPK pathways might play a role in cell proliferation. RNA probes for conserved regions of the catalytic domain of plant MAPK homologue genes were used to study MAPK expression in those plant cells. In situ hybridization (ISH) showed the presence of MAPK transcripts in the three plant cell types studied, but levels were very low in quiescent cells compared to those in cycling cells. The quantification of labelling density of ISH signals strongly suggests a higher level of MAPK expression in proliferating cells, but also some basal messenger presence and/or expression in the quiescent ones. Immunogold and ISH results show the presence and distribution of MAPK proteins and mRNAs in vacuolate microspores. This represents a very dynamic stage during pollen development in which the cell nucleus is being prepared for an asymmetrical mitotic division, giving rise to both the generative and the vegetative nuclei of the bicellular pollen grain. Taken together, the data indicate a role played by MAPK in the re-entry into proliferation in plant cells.

Identification and preliminary characterization of a 65 kDa higher-plant microtubule-associated protein

1993 ◽  
Vol 105 (4) ◽  
pp. 891-901 ◽  
Author(s):  
J. Chang-Jie ◽  
S. Sonobe
Keyword(s):  
Polyclonal Antibodies ◽  
Cold Treatment ◽  
Plant Cells ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
High Concentration ◽  
Animal Cells ◽  
Higher Plant ◽  
Microtubule Protein

Microtubules in plant cells, as in animal cells, are dynamic structures. However, our lack of knowledge about the constituents of microtubules in plant cells has prevented us from understanding the mechanisms that control microtubule dynamics. To characterize some of these constituents, a cytoplasmic extract was prepared from evacuolated protoplasts (miniprotoplasts) of tobacco BY-2 cells, and microtubules were assembled in the presence of taxol and disassembled by cold treatment in the presence of Ca2+ and a high concentration of NaCl. SDS-PAGE analysis of triple-cycled microtubule protein revealed the presence of 120 kDa, 110 kDa and a group of 60–65 kDa polypeptides in addition to tubulin. Since these polypeptides had copolymerized with tubulin, through the three cycles of assembly and disassembly, and they bundle microtubules, we tentatively identified the three polypeptides as microtubule-associated proteins (MAPs). To characterize these factors further, triple-cycled microtubule protein was fractionated by Mono-Q anion-exchange chromatography and the microtubule-bundling activity of each fraction was examined. Fractions having microtubule-bundling activity contained only the 65 kDa MAP, an indication that the 65 kDa MAP is responsible for the bundling of microtubules. Purified 65 kDa MAP formed cross-bridge structures between adjacent microtubules in vitro. Polyclonal antibodies were raised in mice against the 65 kDa MAP. Immunofluorescence microscopy revealed that the 65 kDa MAP colocalized with microtubules in BY-2 cells throughout the cell cycle. Western blotting analysis of extracts from several species of plants suggested that the 65 kDa MAP and/or related peptides are widely distributed in the plant kingdom.

403 IDENTIFICATION AND CHARACTERIZATION OF A NOVEL MOUSE AND HUMAN MOPT GENE CONTAINING MORN MOTIF PROTEIN IN TESTIS

2007 ◽  
Vol 19 (1) ◽  
pp. 317
Author(s):  
K. M. Kim ◽  
Y.-J. Choi ◽  
H. Song ◽  
K.-C. Hwang ◽  
S.-J. Kang ◽  
...  
Keyword(s):  
Cell Types ◽  
Immunogold Electron Microscopy ◽  
Screening Methods ◽  
Positive Staining ◽  
Oocyte Activation ◽  
Protein Distribution ◽  
Dynamic Regulation ◽  
Theoretical Molecular Mass ◽  
Morn Motif

By subtraction screening methods, we identified a novel mouse and its counterpart, human MOPT gene. The mouse and human MOT gene is localized on mouse chromosome 17E3 and human chromosome 2p22, and spans approximately 7 kb. Analysis of the mouse Mopt sequence revealed the existence of an ORF of 240 bp encoding a putative protein of 79aa amino acids. The predicted protein has a theoretical molecular mass of 8.89 kDa and a calculated isoelectric point of 5.82. The protein was unique; it did not show any similarities with other known protein except for Morn motif domain. Real-time reverse transcriptase polymerase chain reaction and Northern blot analysis revealed that mouse Mopt transcripts are highly and specifically expressed in adult testis and skeletal muscle. In situ hybridization and immunohistochemistry studies showed that mouse Mopt transcript and protein was confined mainly to round and elongated spermatids, except for a few individual dispersed spermatocytes, and increases in abundance in subsequent stages. To characterize Mopt functions in spermiogenesis, we examined Mopt protein distribution in late spermiogenesis by using immunogold electron microscopy: Mopt protein first appeared in the proacrosomic vesicles of the early Golgi phase spermatids. In the final step of spermiogenesis, Mopt expression was translocated from the head cap of an elongated spermatid to the nucleus of mature spermatozoa. However, no other testicular cell types, including somatic cells, spermatogenic cells, and residual cytoplasm that ultimately is engulfed as residual bodies into Sertoli cells, were found to bear Mopt-positive staining. This observation suggested that Mopt may play an important role in dynamic regulation of acrosome biogenesis during late spermiogenesis and an as yet uncharacterized role in oocyte activation during capacitation, after fertilization, or both.

Behaviour of the nucleolar fibrillar centres in reproducing plant cells

1978 ◽  
Vol 36 (2) ◽  
pp. 210-211
Author(s):  
F. J. Medina ◽  
M.I. Rodrlguez-Garcia ◽  
M.C. Risueño
Keyword(s):  
Plant Cells ◽  
Pollen Grains ◽  
Constant Component ◽  
Plant Tissues ◽  
Reproductive Tissue ◽  
Animal Cells ◽  
Meristematic Cells ◽  
Fibrillar Material ◽  
Nucleolar Organizing Region ◽  
Nucleolar Component

The nucleolar fibrillar centres have been described recently in animal cells and in meristematic plant cells, as a constant component of the nucleolus, corresponding to the nucleolar organizing region (NOR).In order to find out if the nucleolar fibrillar centres represent a general nucleolar component, we have studied the nucleolus in several reproducing plant tissues (megasporocytes and megaspore from Pissum sativum and mono and bicellulate pollen grains from Allium cepa and Scilla non-scripta). In these cells the fibrillar centres have a different morphological behaviour during their development.Female reproductive tissueImmediately before the meiosis, the megasporocyte nucleolus has a compact and rounded aspect. The fibrillar part occupies the centre and the granular part the periphery. Inside the fibrillar part, there are clear areas containing in some cases, a dark core immersed in a grey fibrillar material (Fig. 1). A similar structure was described recently in meristematic cells. In the pachytene stage, when the nucleolus is joined to the nuclear envelope, the fibrillar centres are made up of a homogeneous fibrillar material with a medium electron density.

scholarly journals Mg2+ modulates the activity of hyperpolarization-activated calcium currents in plant cells

2020 ◽  
Author(s):  
Fouad Lemtiri-Chlieh ◽  
Stefan T. Arold ◽  
Chris Gehring
Keyword(s):  
Plant Hormone ◽  
Plant Cells ◽  
Guard Cells ◽  
Cell Types ◽  
Calcium Currents ◽  
Binding Motif ◽  
Animal Cells ◽  
Membrane Hyperpolarization ◽  
Cyclic Nucleotide Gated Channels ◽  
Channel Opening

ABSTRACTHyperpolarization-activated calcium channels (HACCs) are found in the plasma membrane and tonoplast of many plant cell types where they have an important role in Ca2+-dependent signaling. The unusual gating properties of HACCs in plants, i.e., activation by membrane hyperpolarization rather than depolarization, dictates that HACCs are normally open at physiological hyperpolarized resting membrane potentials (the so called pump or P-state), thus, if not regulated, they would be continuously leaking Ca2+ into cells. In guard cells, HACCs are permeable to Ca2+, Ba2+ and Mg2+, activated by H2O2 and the plant hormone abscisic acid (ABA) and their activity is greatly reduced by low amounts of free cytosolic Ca2+ ([Ca2+]Cyt) and hence will close during [Ca2+]Cyt surges. Here we demonstrate that the presence of the commonly used Mg-ATP inside the cell greatly reduces HACC activity especially at voltages ≤ −200 mV and that Mg2+ causes this block. We therefore conclude, firstly, that physiological cytosolic Mg2+ levels affect HACCs gating and that channel opening requires either high negative voltages (≥ −200 mV) and/or displacement of Mg2+ away from the immediate vicinity of the channel. Secondly, based on structural comparisons with Mg2+-sensitive animal inward-rectifying K+ channel, we propose that the likely candidate HACCS described here are cyclic nucleotide gated channels (CNGCs), many of which also contain a conserved di-acidic Mg2+-binding motif within their pores. This conclusion is consistent with the electrophysiological data. Finally, we propose that Mg2+, much like in animal cells, is an important component in Ca2+ signalling and homeostasis in plants.

scholarly journals Liver regeneration: a comparison of in situ hybridization for histone mRNA with bromodeoxyuridine labeling for the detection of S-phase cells.

1994 ◽  
Vol 42 (12) ◽  
pp. 1603-1608 ◽  
Author(s):  
M Alison ◽  
Z Chaudry ◽  
J Baker ◽  
I Lauder ◽  
H Pringle
Keyword(s):  
In Situ Hybridization ◽  
Cell Types ◽  
S Phase ◽  
Hybridization Technique ◽  
Spatial And Temporal Patterns ◽  
Animal Cells ◽  
Histone Mrna ◽  
Liver Remnant ◽  
Reliable Marker

We developed an in situ hybridization technique for measurement of proliferative cell numbers through detection of histone mRNA in routinely fixed, paraffin-embedded tissue sections. Histone gene expression is coordinated with the cell cycle, and the increase in expression during S-phase permits unambiguous identification of cells undergoing DNA replication. Histone mRNAs were identified in routinely processed rat liver tissue by non-isotopic in situ hybridization with digoxigenin-labeled oligonucleotide probes. Specific hybrids were detected with alkaline phosphatase-labeled anti-digoxigenin antibody and visualized by BCIP-nitroblue tetrazolium indicator substrate. Unequivocal cytoplasmic labeling was observed in various cell types in the liver remnant during the first 72 hr after a two-thirds partial hepatectomy. The spatial and temporal patterns of histone labeling were almost identical to those obtained by staining with an antibody to bromodeoxyuridine. The identification of histone mRNA appears to be a reliable marker of the S-phase fraction, a technique with the further advantage that the tissue does not have to be first exposed to a nucleotide analogue. Hence, retrospective studies are possible. The probes can be applied to human and animal cells and tissues because the nucleotide sequences of histone genes are conserved.

Freeze-substitution of rye grass and ragweed pollen grains

1990 ◽  
Vol 48 (3) ◽  
pp. 686-687
Author(s):  
Liza B. Martinez ◽  
Susan M. Wick
Keyword(s):  
Cell Function ◽  
Pollen Grains ◽  
Freeze Substitution ◽  
Cell Types ◽  
Rapid Freezing ◽  
Rye Grass ◽  
Acrylic Resins ◽  
Allergenic Proteins ◽  
Cold Embedding ◽  
Structural Preservation

Rapid freezing and freeze-substitution have been employed as alternatives to chemical fixation because of the improved structural preservation obtained in various cell types. This has been attributed to biomolecular immobilization derived from the extremely rapid arrest of cell function. These methods allow the elimination of conventionally used fixatives, which may have denaturing or “masking” effects on proteins. Thus, this makes them ideal techniques for immunocytochemistry, in which preservation of both ultrastructure and antigenicity are important. These procedures are also compatible with cold embedding acrylic resins which are known to increase sensitivity in immunolabelling.This study reveals how rapid freezing and freeze-substitution may prove to be useful in the study of the mobile allergenic proteins of rye grass and ragweed. Most studies have relied on the use of osmium tetroxide to achieve the necessary ultrastructural detail in pollen whereas those that omitted it have had to contend with poor overall preservation.

Characterization of an Intermediate Filament Protein from the Platyhelminth, Dugesia japonica

2020 ◽  
Vol 27 (5) ◽  
pp. 432-446
Author(s):  
Akiko Yamamoto ◽  
Ken-ichiro Matsunaga ◽  
Toyoaki Anai ◽  
Hitoshi Kawano ◽  
Toshihisa Ueda ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Immunohistochemical Analysis ◽  
Protein Characterization ◽  
Intermediate Filament Protein ◽  
Tail Domain ◽  
Animal Cells ◽  
Dugesia Japonica ◽  
Function Relationship

Background: Intermediate Filaments (IFs) are major constituents of the cytoskeletal systems in animal cells. Objective: To gain insights into the structure-function relationship of invertebrate cytoplasmic IF proteins, we characterized an IF protein from the platyhelminth, Dugesia japonica, termed Dif-1. Method: cDNA cloning, in situ hybridization, immunohistochemical analysis, and IF assembly experiments in vitro using recombinant Dif-1, were performed for protein characterization. Results: The structure deduced from the cDNA sequence showed that Djf-1 comprises 568 amino acids and has a tripartite domain structure (N-terminal head, central rod, and C-terminal tail) that is characteristic of IF proteins. Similar to nuclear IF lamins, Djf-1 contains an extra 42 residues in the coil 1b subdomain of the rod domain that is absent from vertebrate cytoplasmic IF proteins and a nuclear lamin-homology segment of approximately 105 residues in the tail domain; however, it contains no nuclear localization signal. In situ hybridization analysis showed that Djf-1 mRNA is specifically expressed in cells located within the marginal region encircling the worm body. Immunohistochemical analysis showed that Djf-1 protein forms cytoplasmic IFs located close to the microvilli of the cells. In vitro IF assembly experiments using recombinant proteins showed that Djf-1 alone polymerizes into IFs. Deletion of the extra 42 residues in the coil 1b subdomain resulted in the failure of IF formation. Conclusions: Together with data from other histological studies, our results suggest that Djf- 1 is expressed specifically in anchor cells within the glandular adhesive organs of the worm and that Djf-1 IFs may play a role in protecting the cells from mechanical stress.

scholarly journals In situ differentiation of iridophore crystallotypes underlies zebrafish stripe patterning

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Dvir Gur ◽  
Emily J. Bain ◽  
Kory R. Johnson ◽  
Andy J. Aman ◽  
H. Amalia Pasoili ◽  
...  
Keyword(s):  
Skin Color ◽  
Cell Types ◽  
Color Pattern ◽  
Single Type ◽  
Sequencing Analysis ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cryogenic Scanning Electron Microscopy ◽  
Different Cell Types

AbstractSkin color patterns are ubiquitous in nature, impact social behavior, predator avoidance, and protection from ultraviolet irradiation. A leading model system for vertebrate skin patterning is the zebrafish; its alternating blue stripes and yellow interstripes depend on light-reflecting cells called iridophores. It was suggested that the zebrafish’s color pattern arises from a single type of iridophore migrating differentially to stripes and interstripes. However, here we find that iridophores do not migrate between stripes and interstripes but instead differentiate and proliferate in-place, based on their micro-environment. RNA-sequencing analysis further reveals that stripe and interstripe iridophores have different transcriptomic states, while cryogenic-scanning-electron-microscopy and micro-X-ray diffraction identify different crystal-arrays architectures, indicating that stripe and interstripe iridophores are different cell types. Based on these results, we present an alternative model of skin patterning in zebrafish in which distinct iridophore crystallotypes containing specialized, physiologically responsive, organelles arise in stripe and interstripe by in-situ differentiation.

Pituitary adenoma producing growth hormone and adrenocorticotropin: a histological, immunocytochemical, electron microscopic, and in situ hybridization study

1998 ◽  
Vol 88 (6) ◽  
pp. 1111-1115 ◽  
Author(s):  
Kalman Kovacs ◽  
Eva Horvath ◽  
Lucia Stefaneanu ◽  
Juan Bilbao ◽  
William Singer ◽  
...  
Keyword(s):  
Growth Hormone ◽  
In Situ Hybridization ◽  
Pituitary Adenoma ◽  
Immunoelectron Microscopy ◽  
Secretory Granules ◽  
Cell Types ◽  
Content Type ◽  
Separate Cell ◽  
Fine Print

✓ The authors report on the morphological features of a pituitary adenoma that produced growth hormone (GH) and adrenocorticotropic hormone (ACTH). This hormone combination produced by a single adenoma is extremely rare; a review of the available literature showed that only one previous case has been published. The tumor, which was removed from a 62-year-old man with acromegaly, was studied by histological and immunocytochemical analyses, transmission electron microscopy, immunoelectron microscopy, and in situ hybridization. When the authors used light microscopy, the tumor appeared to be a bimorphous mixed pituitary adenoma composed of two separate cell types: one cell population synthesized GH and the other ACTH. The cytogenesis of pituitary adenomas that produce more than one hormone is obscure. It may be that two separate cells—one somatotroph and one corticotroph—transformed into neoplastic cells, or that the adenoma arose in a common stem cell that differentiated into two separate cell types. In this case immunoelectron microscopy conclusively demonstrated ACTH in the secretory granules of several somatotrophs. This was associated with a change in the morphological characteristics of secretory granules. Thus it is possible that the tumor was originally a somatotropic adenoma that began to produce ACTH as a result of mutations that occurred during tumor progression.

Application of YO-PRO-1 as an Epifluorescent Dye for in situ Detection of Small Amount DNA in Plant Cells

1999 ◽  
Vol 112 (1) ◽  
pp. 117-122 ◽  
Author(s):  
Sodmergen ◽  
Jian-Gong Niu ◽  
Lin-Jiang Li ◽  
Jiang-Xun He ◽  
Feng-Li Guo
Keyword(s):  
Plant Cells ◽  
In Situ Detection
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