scholarly journals Dynamic elastic behavior of alpha-satellite DNA domains visualized in situ in living human cells.

1996 ◽  
Vol 135 (3) ◽  
pp. 545-557 ◽  
Author(s):  
R D Shelby ◽  
K M Hahn ◽  
K F Sullivan
Keyword(s):  
Satellite Dna ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Fluorescent Labeling ◽  
Human Cells ◽  
Elastic Behavior ◽  
Alpha Satellite ◽  
Alpha Satellite Dna ◽  
Green Fluorescent

We have constructed a fluorescent alpha-satellite DNA-binding protein to explore the motile and mechanical properties of human centromeres. A fusion protein consisting of human CENP-B coupled to the green fluorescent protein (GFP) of A. victoria specifically targets to centromeres when expressed in human cells. Morphometric analysis revealed that the alpha-satellite DNA domain bound by CENPB-GFP becomes elongated in mitosis in a microtubule-dependent fashion. Time lapse confocal microscopy in live mitotic cells revealed apparent elastic deformations of the central domain of the centromere that occurred during metaphase chromosome oscillations. These observations demonstrate that the interior region of the centromere behaves as an elastic element that could play a role in the mechanoregulatory mechanisms recently identified at centromeres. Fluorescent labeling of centromeres revealed that they disperse throughout the nucleus in a nearly isometric expansion during chromosome decondensation in telophase and early G1. During interphase, centromeres were primarily stationary, although motility of individual or small groups of centromeres was occasionally observed at very slow rates of 7-10 microns/h.

Detection of an i(17q) chromosome by fluorescent in situ hybridization with a chromosome 17 alpha satellite DNA probe

1992 ◽  
Vol 62 (2) ◽  
pp. 140-143 ◽  
Author(s):  
Hitoshi Nakagawa ◽  
Johji Inazawa ◽  
Shinichi Misawa ◽  
Shinji Tanaka ◽  
Teruyuki Takashima ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Satellite Dna ◽  
Fluorescent In Situ Hybridization ◽  
Dna Probe ◽  
Chromosome 17 ◽  
Alpha Satellite ◽  
Alpha Satellite Dna

Oligonucleotide-priming methods for the chromosome-specific labelling of alpha satellite DNA in situ

Chromosoma ◽  
1989 ◽  
Vol 98 (4) ◽  
pp. 259-265 ◽  
Author(s):  
J�rn E. Koch ◽  
Steen K�lvraa ◽  
Kirsten B. Petersen ◽  
Niels Gregersen ◽  
Lars Bolund
Keyword(s):  
Satellite Dna ◽  
Alpha Satellite ◽  
Alpha Satellite Dna ◽  
Specific Labelling

scholarly journals In Vivo Analysis of Cajal Body Movement, Separation, and Joining in Live Human Cells

2000 ◽  
Vol 151 (7) ◽  
pp. 1561-1574 ◽  
Author(s):  
Melpomeni Platani ◽  
Ilya Goldberg ◽  
Jason R. Swedlow ◽  
Angus I. Lamond
Keyword(s):  
Fluorescent Protein ◽  
Body Movement ◽  
Time Lapse ◽  
Biochemical Properties ◽  
Human Cells ◽  
Nuclear Bodies ◽  
Cajal Body ◽  
Cajal Bodies ◽  
Green Fluorescent

Cajal bodies (also known as coiled bodies) are subnuclear organelles that contain specific nuclear antigens, including splicing small nuclear ribonucleoproteins (snRNPs) and a subset of nucleolar proteins. Cajal bodies are localized in the nucleoplasm and are often found at the nucleolar periphery. We have constructed a stable HeLa cell line, HeLaGFP-coilin, that expresses the Cajal body marker protein, p80 coilin, fused to the green fluorescent protein (GFP-coilin). The localization pattern and biochemical properties of the GFP-coilin fusion protein are identical to the endogenous p80 coilin. Time-lapse recordings on 63 nuclei of HeLaGFP-coilin cells showed that all Cajal bodies move within the nucleoplasm. Movements included translocations through the nucleoplasm, joining of bodies to form larger structures, and separation of smaller bodies from larger Cajal bodies. Also, we observed Cajal bodies moving to and from nucleoli. The data suggest that there may be at least two classes of Cajal bodies that differ in their size, antigen composition, and dynamic behavior. The smaller size class shows more frequent and faster rates of movement, up to 0.9 μm/min. The GFP-coilin protein is dynamically associated with Cajal bodies as shown by changes in their fluorescence intensity over time. This study reveals an unexpectedly high level of movement and interactions of nuclear bodies in human cells and suggests that these movements may be driven, at least in part, by regulated mechanisms.

scholarly journals Mutational Analysis of Fibrillarin and Its Mobility in Living Human Cells

2000 ◽  
Vol 151 (3) ◽  
pp. 653-662 ◽  
Author(s):  
Sabine Snaar ◽  
Karien Wiesmeijer ◽  
Aart G. Jochemsen ◽  
Hans J. Tanke ◽  
Roeland W. Dirks
Keyword(s):  
Fluorescent Protein ◽  
Mutational Analysis ◽  
Diffusion Constant ◽  
Alpha Helix ◽  
Time Lapse ◽  
Human Cells ◽  
Low Frequencies ◽  
Mobile Fraction ◽  
Green Fluorescent ◽  
Carboxy Terminal

Cajal bodies (CBs) are subnuclear organelles that contain components of a number of distinct pathways in RNA transcription and RNA processing. CBs have been linked to other subnuclear organelles such as nucleoli, but the reason for the presence of nucleolar proteins such as fibrillarin in CBs remains uncertain. Here, we use full-length fibrillarin and truncated fibrillarin mutants fused to green fluorescent protein (GFP) to demonstrate that specific structural domains of fibrillarin are required for correct intranuclear localization of fibrillarin to nucleoli and CBs. The second spacer domain and carboxy terminal alpha-helix domain in particular appear to target fibrillarin, respectively, to the nucleolar transcription centers and CBs. The presence of the RNP domain seems to be a prerequisite for correct targeting of fibrillarin. Time-lapse confocal microscopy of human cells that stably express fibrillarin-GFP shows that CBs fuse and split, albeit at low frequencies. Recovered fluorescence of fibrillarin-GFP in nucleoli and CBs after photobleaching indicates that it is highly mobile in both organelles (estimated diffusion constant ∼0.02 μm2 s−1), and has a significantly larger mobile fraction in CBs than in nucleoli.

scholarly journals The Sorting of Mitochondrial DNA and Mitochondrial Proteins in Zygotes: Preferential Transmission of Mitochondrial DNA to the Medial Bud

1998 ◽  
Vol 142 (3) ◽  
pp. 613-623 ◽  
Author(s):  
Koji Okamoto ◽  
Philip S. Perlman ◽  
Ronald A. Butow
Keyword(s):  
Mitochondrial Dna ◽  
Fluorescent Protein ◽  
Yeast Cells ◽  
Mitochondrial Matrix ◽  
Outer Membranes ◽  
Marker Proteins ◽  
The Matrix ◽  
Green Fluorescent ◽  
Mitochondrial Reticulum

Green fluorescent protein (GFP) was used to tag proteins of the mitochondrial matrix, inner, and outer membranes to examine their sorting patterns relative to mtDNA in zygotes of synchronously mated yeast cells in ρ+ × ρ0 crosses. When transiently expressed in one of the haploid parents, each of the marker proteins distributes throughout the fused mitochondrial reticulum of the zygote before equilibration of mtDNA, although the membrane markers equilibrate slower than the matrix marker. A GFP-tagged form of Abf2p, a mtDNA binding protein required for faithful transmission of ρ+ mtDNA in vegetatively growing cells, colocalizes with mtDNA in situ. In zygotes of a ρ+ × ρ+ cross, in which there is little mixing of parental mtDNAs, Abf2p–GFP prelabeled in one parent rapidly equilibrates to most or all of the mtDNA, showing that the mtDNA compartment is accessible to exchange of proteins. In ρ+ × ρ0 crosses, mtDNA is preferentially transmitted to the medial diploid bud, whereas mitochondrial GFP marker proteins distribute throughout the zygote and the bud. In zygotes lacking Abf2p, mtDNA sorting is delayed and preferential sorting is reduced. These findings argue for the existence of a segregation apparatus that directs mtDNA to the emerging bud.

scholarly journals Imaging and Analysis of Pseudomonas aeruginosa Swarming and Rhamnolipid Production

2011 ◽  
Vol 77 (23) ◽  
pp. 8310-8317 ◽  
Author(s):  
Joshua D. Morris ◽  
Jessica L. Hewitt ◽  
Lawrence G. Wolfe ◽  
Nachiket G. Kamatkar ◽  
Sarah M. Chapman ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fluorescent Protein ◽  
Nile Red ◽  
Temporal Distribution ◽  
Time Lapse ◽  
Content Type ◽  
Rhamnolipid Production ◽  
Serovar Typhimurium ◽  
Surface Motility ◽  
Green Fluorescent

ABSTRACTMany bacteria spread over surfaces by “swarming” in groups. A problem for scientists who study swarming is the acquisition of statistically significant data that distinguish two observations or detail the temporal patterns and two-dimensional heterogeneities that occur. It is currently difficult to quantify differences between observed swarm phenotypes. Here, we present a method for acquisition of temporal surface motility data using time-lapse fluorescence and bioluminescence imaging. We specifically demonstrate three applications of our technique with the bacteriumPseudomonas aeruginosa. First, we quantify the temporal distribution ofP. aeruginosacells tagged with green fluorescent protein (GFP) and the surfactant rhamnolipid stained with the lipid dye Nile red. Second, we distinguish swarming ofP. aeruginosaandSalmonella entericaserovar Typhimurium in a coswarming experiment. Lastly, we quantify differences in swarming and rhamnolipid production of severalP. aeruginosastrains. While the best swarming strains produced the most rhamnolipid on surfaces, planktonic culture rhamnolipid production did not correlate with surface growth rhamnolipid production.

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