scholarly journals In vivo veritas: the power of in situ manipulation of cells in a living animal. Focus on “Expression of plasmid DNA in the salivary gland epithelium: novel approaches to study dynamic cellular processes in live animals”

2009 ◽  
Vol 297 (6) ◽  
pp. C1333-C1335 ◽  
Author(s):  
Bruce J. Baum
Keyword(s):  
Salivary Gland ◽  
Plasmid Dna ◽  
Living Animal ◽  
Cellular Processes ◽  
Approaches To Study ◽  
Novel Approaches

scholarly journals Expression of plasmid DNA in the salivary gland epithelium: novel approaches to study dynamic cellular processes in live animals

2009 ◽  
Vol 297 (6) ◽  
pp. C1347-C1357 ◽  
Author(s):  
Monika Sramkova ◽  
Andrius Masedunskas ◽  
Laura Parente ◽  
Alfredo Molinolo ◽  
Roberto Weigert
Keyword(s):  
Plasmid Dna ◽  
Target Genes ◽  
Specific Cell ◽  
Experimental Conditions ◽  
Live Animal ◽  
Cellular Processes ◽  
Submandibular Salivary Glands ◽  
Powerful Approach ◽  
Approaches To Study ◽  
Granular Ducts

The ability to dynamically image cellular and subcellular structures in a live animal and to target genes to a specific cell population in a living tissue provides a unique tool to address many biological questions in the proper physiological context. Here, we describe a powerful approach that is based on the use of rat submandibular salivary glands, which offer the possibility to easily perform intravital imaging and deliver molecules from the oral cavity, and plasmid DNA, which offers the advantage of rapid manipulations. We show that, under different experimental conditions, a reporter molecule can be rapidly expressed in specific compartments of the glands: 1) in the intercalated ducts, when plasmid DNA is administered alone, and 2) in granular ducts, striated ducts, and, to a lesser extent, acini, when plasmid DNA is mixed with replication-deficient adenovirus subtype 5 particles. Remarkably, we also found that gene expression can be directed to acinar cells when plasmid DNA is administered during isoproterenol-stimulated exocytosis, suggesting a novel mechanism of plasmid internalization regulated by compensatory endocytosis. Finally, as a practical application of these strategies, we show how the expression of fluorescently tagged molecules enables the study of the dynamics of various organelles in live animals at a resolution comparable to that achieved in cell cultures.

scholarly journals Chromatin is frequently unknotted at the megabase scale

2019 ◽  
Author(s):  
Dimos Goundaroulis ◽  
Erez Lieberman Aiden ◽  
Andrzej Stasiak
Keyword(s):  
Gene Regulation ◽  
Human Genome ◽  
Single Locus ◽  
Chromosome 21 ◽  
Cellular Processes ◽  
Potential Source ◽  
Localization Errors

Knots in the human genome would greatly impact diverse cellular processes ranging from transcription to gene regulation. To date, it has not been possible to directly examine the genome in vivo for the presence of knots. Recently, methods for serial fluorescent in situ hybridization have made it possible to measure the 3d position of dozens of consecutive genomic loci, in vivo. However, the determination of whether genomic trajectories are knotted remains challenging, because small errors in the localization of a single locus can transform an unknotted trajectory into a highly-knotted trajectory, and vice versa. Here, we use stochastic closure analysis to determine whether a genomic trajectory is knotted in the setting of experimental noise. We analyse 4727 deposited genomic trajectories of a 2Mb long chromatin interval from chromosome 21. For 243 of these trajectories, their knottedness could be reliably determined despite the possibility of localization errors. Strikingly, in each of these 243 cases, the trajectory was unknotted. We note a potential source of bias, insofar as knotted contours may be more difficult to reliably resolve. Nevertheless, our data is consistent with a model where, at the scales probed, the human genome is often free of knots.

scholarly journals Relative Expression Levels Rather Than Specific Activity Plays the Major Role in Determining In Vivo AKT Isoform Substrate Specificity

2011 ◽  
Vol 2011 ◽  
pp. 1-18 ◽  
Author(s):  
Rachel S. Lee ◽  
Colin M. House ◽  
Briony E. Cristiano ◽  
Ross D. Hannan ◽  
Richard B. Pearson ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Specific Activity ◽  
Dominant Role ◽  
Protein Substrates ◽  
Cellular Processes ◽  
Disease States ◽  
The Individual

The AKT protooncogene mediates many cellular processes involved in normal development and disease states such as cancer. The three structurally similar isoforms: AKT1, AKT2, and AKT3 exhibit both functional redundancy and isoform-specific functions; however the basis for their differential signalling remains unclear. Here we show that in vitro, purified AKT3 is ∼47-fold more active than AKT1 at phosphorylating peptide and protein substrates. Despite these marked variations in specific activity between the individual isoforms, a comprehensive analysis of phosphorylation of validated AKT substrates indicated only subtle differences in signalling via individual isoforms in vivo. Therefore, we hypothesise, at least in this model system, that relative tissue/cellular abundance, rather than specific activity, plays the dominant role in determining AKT substrate specificity in situ.

scholarly journals The Drosophila salivary gland chromocenter contains highly polytenized subdomains of mitotic heterochromatin.

Genetics ◽  
1995 ◽  
Vol 139 (2) ◽  
pp. 659-670 ◽  
Author(s):  
P Zhang ◽  
A C Spradling
Keyword(s):  
Salivary Gland ◽  
Dna Sequences ◽  
Polytene Chromosomes ◽  
Centromeric Heterochromatin ◽  
Mitotic Chromosomes ◽  
Satellite Dnas ◽  
Central Mass ◽  
P Elements

Abstract Peri-centromeric regions of Drosophila melanogaster chromosomes appear heterochromatic in mitotic cells and become greatly underrepresented in giant polytene chromosomes, where they aggregate into a central mass called the chromocenter. We used P elements inserted at sites dispersed throughout much of the mitotic heterochromatin to analyze the fate of 31 individual sites during polytenization. Analysis of DNA sequences flanking many of these elements revealed that middle repetitive or unique sequence DNAs frequently are interspersed with satellite DNAs in mitotic heterochromatin. All nine Y chromosome sites tested were underrepresented > 20-fold on Southern blots of polytene DNA and were rarely or never detected by in situ hybridization to salivary gland chromosomes. In contrast, nine tested insertions in autosomal centromeric heterochromatin were represented fully in salivary gland DNA, despite the fact that at least six were located proximal to known blocks of satellite DNA. The inserted sequences formed diverse, site-specific morphologies in the chromocenter of salivary gland chromosomes, suggesting that domains dispersed at multiple sites in the centromeric heterochromatin of mitotic chromosomes contribute to polytene beta-heterochromatin. We suggest that regions containing heterochromatic genes are organized into dispersed chromatin configurations that are important for their function in vivo.

Identification and taxonomy of soft-bodied hexacorals exemplified by Chilean sea anemones; including guidelines for sampling, preservation and examination

2004 ◽  
Vol 84 (5) ◽  
pp. 931-936 ◽  
Author(s):  
Verena Häussermann
Keyword(s):  
Sampling Methods ◽  
Sea Anemone ◽  
Living Animal ◽  
Correct Interpretation ◽  
Sea Anemones ◽  
Ecological Information ◽  
Damaged Material ◽  
Species Descriptions

The identification of most soft-bodied hexacorals requires morphological and histological examinations of preserved specimens and experience for correct interpretation of the observed features. Poorly preserved or damaged material resulting from improper sampling complicates identification. In many cases the characteristics of the preserved specimens alone do not lead to satisfying results. Living specimens, however, exhibit numerous characteristics which would often allow identification, even in the field. However, most of these characteristics get lost during preservation. Modern techniques and advances in sampling methods allow the acquisition and preservation of a lot of information on the living animal and its habitat.Using Chilean sea anemone species, it is demonstrated how the work with specimens in situ and in vivo can help with identification and reveal important morphological–taxonomical, biological, and ecological information. Whenever possible, this information should be part of species descriptions and should be used to create detailed, reliable, tabular identification keys for the laboratory and field. The examples illustrate the urgent need for modern, comparable re-descriptions. In most parts the protocol also applies to other soft-bodied hexacorals.

scholarly journals In Situ Transcriptome Accessibility Sequencing (INSTA-seq)

2019 ◽  
Author(s):  
Daniel Fürth ◽  
Victor Hatini ◽  
Je H. Lee
Keyword(s):  
Protein Interactions ◽  
Rna Localization ◽  
Cytoskeletal Organization ◽  
Short Reads ◽  
Animal Development ◽  
Cellular Processes ◽  
Regulatory Variants ◽  
Sequencing Chemistry

Subcellular RNA localization regulates spatially polarized cellular processes, but unbiased investigation of its control in vivo remains challenging. Current hybridization-based methods cannot differentiate small regulatory variants, while in situ sequencing is limited by short reads. We solved these problems using a bidirectional sequencing chemistry to efficiently image transcript-specific barcode in situ, which are then extracted and assembled into longer reads using NGS. In the Drosophila retina, genes regulating eye development and cytoskeletal organization were enriched compared to methods using extracted RNA. We therefore named our method In Situ Transcriptome Accessibility sequencing (INSTA-seq). Sequencing reads terminated near 3’ UTR cis-motifs (e.g. Zip48C, stau), revealing RNA-protein interactions. Additionally, Act5C polyadenylation isoforms retaining zipcode motifs were selectively localized to the optical stalk, consistent with their biology. Our platform provides a powerful way to visualize any RNA variants or protein interactions in situ to study their regulation in animal development.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

Three-Dimensional Subcellular Organization of Hepatocytes in Intact Liver: Simultaneous Visualization of Cytoskeletal and Membrane Markers by Confocal Microscopy

1996 ◽  
Vol 54 ◽  
pp. 288-289
Author(s):  
Greg V. Martin ◽  
Ann L. Hubbard
Keyword(s):  
Three Dimensional ◽  
Integral Membrane Proteins ◽  
Polarized Secretion ◽  
Microscope Images ◽  
Computer Aided ◽  
Intracellular Organelles ◽  
Cytoskeletal Elements ◽  
Simultaneous Visualization

The microtubule (MT) cytoskeleton is necessary for many of the polarized functions of hepatocytes. Among the functions dependent on the MT-based cytoskeleton are polarized secretion of proteins, delivery of endocytosed material to lysosomes, and transcytosis of integral plasma membrane (PM) proteins. Although microtubules have been shown to be crucial to the establishment and maintenance of functional and structural polarization in the hepatocyte, little is known about the architecture of the hepatocyte MT cytoskeleton in vivo, particularly with regard to its relationship to PM domains and membranous organelles. Using an in situ extraction technique that preserves both microtubules and cellular membranes, we have developed a protocol for immunofluorescent co-localization of cytoskeletal elements and integral membrane proteins within 20 µm cryosections of fixed rat liver. Computer-aided 3D reconstruction of multi-spectral confocal microscope images was used to visualize the spatial relationships among the MT cytoskeleton, PM domains and intracellular organelles.

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