scholarly journals The Dynamics of Postmitotic Reassembly of the Nucleolus

2000 ◽  
Vol 150 (3) ◽  
pp. 433-446 ◽  
Author(s):  
Miroslav Dundr ◽  
Tom Misteli ◽  
Mark O.J. Olson
Keyword(s):  
Fluorescence Recovery After Photobleaching ◽  
Fluorescent Protein ◽  
Living Cells ◽  
M Phase ◽  
Specific Proteins ◽  
Distribution Of Components ◽  
Green Fluorescent ◽  
Related Proteins ◽  
First Time ◽  
Rrna Sequences

Mammalian cell nucleoli disassemble at the onset of M-phase and reassemble during telophase. Recent studies showed that partially processed preribosomal RNA (pre-rRNA) is preserved in association with processing components in the perichromosomal regions (PRs) and in particles called nucleolus-derived foci (NDF) during mitosis. Here, the dynamics of nucleolar reassembly were examined for the first time in living cells expressing fusions of the processing-related proteins fibrillarin, nucleolin, or B23 with green fluorescent protein (GFP). During telophase the NDF disappeared with a concomitant appearance of material in the reforming nuclei. Prenucleolar bodies (PNBs) appeared in nuclei in early telophase and gradually disappeared as nucleoli formed, strongly suggesting the transfer of PNB components to newly forming nucleoli. Fluorescence recovery after photobleaching (FRAP) showed that fibrillarin-GFP reassociates with the NDF and PNBs at rapid and similar rates. The reentry of processing complexes into telophase nuclei is suggested by the presence of pre-rRNA sequences in PNBs. Entry of specific proteins into the nucleolus approximately correlated with the timing of processing events. The mitotically preserved processing complexes may be essential for regulating the distribution of components to reassembling daughter cell nucleoli.

scholarly journals Motile Properties of Vimentin Intermediate Filament Networks in Living Cells

1998 ◽  
Vol 143 (1) ◽  
pp. 147-157 ◽  
Author(s):  
Miri Yoon ◽  
Robert D. Moir ◽  
Veena Prahlad ◽  
Robert D. Goldman
Keyword(s):  
Intermediate Filament ◽  
Cell Shape ◽  
Fluorescence Recovery After Photobleaching ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Living Cells ◽  
A Cell ◽  
Filament Networks ◽  
Green Fluorescent ◽  
Vimentin Intermediate Filament

The motile properties of intermediate filament (IF) networks have been studied in living cells expressing vimentin tagged with green fluorescent protein (GFP-vimentin). In interphase and mitotic cells, GFP-vimentin is incorporated into the endogenous IF network, and accurately reports the behavior of IF. Time-lapse observations of interphase arrays of vimentin fibrils demonstrate that they are constantly changing their configurations in the absence of alterations in cell shape. Intersecting points of vimentin fibrils, or foci, frequently move towards or away from each other, indicating that the fibrils can lengthen or shorten. Fluorescence recovery after photobleaching shows that bleach zones across fibrils rapidly recover their fluorescence. During this recovery, bleached zones frequently move, indicating translocation of fibrils. Intriguingly, neighboring fibrils within a cell can exhibit different rates and directions of movement, and they often appear to extend or elongate into the peripheral regions of the cytoplasm. In these same regions, short filamentous structures are also seen actively translocating. All of these motile properties require energy, and the majority appear to be mediated by interactions of IF with microtubules and microfilaments.

scholarly journals Rhinovirus 3C Protease Can Localize in the Nucleus and Alter Active and Passive Nucleocytoplasmic Transport

2009 ◽  
Vol 83 (14) ◽  
pp. 7349-7352 ◽  
Author(s):  
Reena Ghildyal ◽  
Benjamin Jordan ◽  
Dongsheng Li ◽  
Hayat Dagher ◽  
Phillip G. Bardin ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Intact Cell ◽  
Nucleocytoplasmic Transport ◽  
Living Cells ◽  
Nuclear Pore ◽  
Nucleocytoplasmic Trafficking ◽  
3C Protease ◽  
Infected Cells ◽  
Green Fluorescent ◽  
First Time

ABSTRACT The degradation of nuclear pore components and disruption of nucleocytoplasmic trafficking during rhinovirus infection have been attributed to viral 2A protease. Here we show for the first time that rhinovirus 3C protease may also have a role. Specifically, we show that 3C and its precursor, 3CD, can target green fluorescent protein to the nucleus of living cells, leading to degradation of nuclear pore components, and that incubation with recombinant 3C disrupts active and passive nucleocytoplasmic transport in a semi-intact cell nuclear transport system dependent on 3C protease activity. 3C may thus contribute to host cell shutoff in infected cells by localizing in the nucleus and facilitating nuclear pore breakdown.

scholarly journals Direct Visualization of the Translocation of the γ-Subspecies of Protein Kinase C in Living Cells Using Fusion Proteins with Green Fluorescent Protein

1997 ◽  
Vol 139 (6) ◽  
pp. 1465-1476 ◽  
Author(s):  
Norio Sakai ◽  
Keiko Sasaki ◽  
Natsu Ikegaki ◽  
Yasuhito Shirai ◽  
Yoshitaka Ono ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Green Fluorescent Protein ◽  
Nmda Receptor ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
Living Cells ◽  
Kinase C ◽  
Gfp Fusion Protein ◽  
Green Fluorescent

We expressed the γ-subspecies of protein kinase C (γ-PKC) fused with green fluorescent protein (GFP) in various cell lines and observed the movement of this fusion protein in living cells under a confocal laser scanning fluorescent microscope. γ-PKC–GFP fusion protein had enzymological properties very similar to that of native γ-PKC. The fluorescence of γ-PKC– GFP was observed throughout the cytoplasm in transiently transfected COS-7 cells. Stimulation by an active phorbol ester (12-O-tetradecanoylphorbol 13-acetate [TPA]) but not by an inactive phorbol ester (4α-phorbol 12, 13-didecanoate) induced a significant translocation of γ-PKC–GFP from cytoplasm to the plasma membrane. A23187, a Ca2+ ionophore, induced a more rapid translocation of γ-PKC–GFP than TPA. The A23187-induced translocation was abolished by elimination of extracellular and intracellular Ca2+. TPA- induced translocation of γ-PKC–GFP was unidirected, while Ca2+ ionophore–induced translocation was reversible; that is, γ-PKC–GFP translocated to the membrane returned to the cytosol and finally accumulated as patchy dots on the plasma membrane. To investigate the significance of C1 and C2 domains of γ-PKC in translocation, we expressed mutant γ-PKC–GFP fusion protein in which the two cysteine rich regions in the C1 region were disrupted (designated as BS 238) or the C2 region was deleted (BS 239). BS 238 mutant was translocated by Ca2+ ionophore but not by TPA. In contrast, BS 239 mutant was translocated by TPA but not by Ca2+ ionophore. To examine the translocation of γ-PKC–GFP under physiological conditions, we expressed it in NG-108 cells, N-methyl-d-aspartate (NMDA) receptor–transfected COS-7 cells, or CHO cells expressing metabotropic glutamate receptor 1 (CHO/mGluR1 cells). In NG-108 cells , K+ depolarization induced rapid translocation of γ-PKC–GFP. In NMDA receptor–transfected COS-7 cells, application of NMDA plus glycine also translocated γ-PKC–GFP. Furthermore, rapid translocation and sequential retranslocation of γ-PKC–GFP were observed in CHO/ mGluR1 cells on stimulation with the receptor. Neither cytochalasin D nor colchicine affected the translocation of γ-PKC–GFP, indicating that translocation of γ-PKC was independent of actin and microtubule. γ-PKC–GFP fusion protein is a useful tool for investigating the molecular mechanism of γ-PKC translocation and the role of γ-PKC in the central nervous system.

Mammalian cell lines expressing functional RNA polymerase II tagged with the green fluorescent protein

2000 ◽  
Vol 113 (15) ◽  
pp. 2679-2683 ◽  
Author(s):  
K. Sugaya ◽  
M. Vigneron ◽  
P.R. Cook
Keyword(s):  
Green Fluorescent Protein ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Living Cells ◽  
Temperature Sensitive ◽  
Mammalian Cell Lines ◽  
Eukaryotic Genes ◽  
Green Fluorescent

RNA polymerase II is a multi-subunit enzyme responsible for transcription of most eukaryotic genes. It associates with other complexes to form enormous multifunctional ‘holoenzymes’ involved in splicing and polyadenylation. We wished to study these different complexes in living cells, so we generated cell lines expressing the largest, catalytic, subunit of the polymerase tagged with the green fluorescent protein. The tagged enzyme complements a deficiency in tsTM4 cells that have a temperature-sensitive mutation in the largest subunit. Some of the tagged subunit is incorporated into engaged transcription complexes like the wild-type protein; it both resists extraction with sarkosyl and is hyperphosphorylated at its C terminus. Remarkably, subunits bearing such a tag can be incorporated into the active enzyme, despite the size and complexity of the polymerizing complex. Therefore, these cells should prove useful in the analysis of the dynamics of transcription in living cells.

scholarly journals Generation of iPSCs from Jaw Periosteal Cells Using Self-Replicating RNA

2019 ◽  
Vol 20 (7) ◽  
pp. 1648 ◽  
Author(s):  
Felix Umrath ◽  
Heidrun Steinle ◽  
Marbod Weber ◽  
Hans-Peter Wendel ◽  
Siegmar Reinert ◽  
...  
Keyword(s):  
Stem Cells ◽  
Fluorescent Protein ◽  
Osteogenic Potential ◽  
Safety Standards ◽  
Stem Cell Source ◽  
Periosteal Cells ◽  
Induced Pluripotent ◽  
Green Fluorescent ◽  
First Time

Jaw periosteal cells (JPCs) represent a suitable stem cell source for bone tissue engineering (BTE) applications. However, challenges associated with limited cell numbers, stressful cell sorting, or the occurrence of cell senescence during in vitro passaging and the associated insufficient osteogenic potential in vitro of JPCs and other mesenchymal stem/stromal cells (MSCs) are main hurdles and still need to be solved. In this study, for the first time, induced pluripotent stem cells (iPSCs) were generated from human JPCs to open up a new source of stem cells for BTE. For this purpose, a non-integrating self-replicating RNA (srRNA) encoding reprogramming factors and green fluorescent protein (GFP) as a reporter was used to obtain JPC-iPSCs with a feeder- and xeno-free reprogramming protocol to meet the highest safety standards for future clinical applications. Furthermore, to analyze the potential of these iPSCs as a source of osteogenic progenitor cells, JPC-iPSCs were differentiated into iPSC-derived mesenchymal stem/stromal like cells (iMSCs) and further differentiated to the osteogenic lineage under xeno-free conditions. The produced iMSCs displayed MSC marker expression and morphology as well as strong mineralization during osteogenic differentiation.

scholarly journals Stable hZW10 kinetochore residency, mediated by hZwint-1 interaction, is essential for the mitotic checkpoint

2008 ◽  
Vol 180 (3) ◽  
pp. 507-520 ◽  
Author(s):  
Jakub K. Famulski ◽  
Larissa Vos ◽  
Xuejun Sun ◽  
Gordon Chan
Keyword(s):  
Chromosome Segregation ◽  
Fluorescence Recovery After Photobleaching ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Mitotic Checkpoint ◽  
Interaction Domain ◽  
Mutagenesis Screen ◽  
Localization Domain ◽  
Green Fluorescent ◽  
Surveillance Mechanism

The mitotic checkpoint is an essential surveillance mechanism that ensures high fidelity chromosome segregation during mitosis. Mitotic checkpoint function depends on numerous kinetochore proteins, including ZW10, ROD, and Zwilch (the ROD–ZW10–Zwilch complex). Through an extensive mutagenesis screen of hZW10, we have mapped the kinetochore localization domain of hZW10 as well as the hZwint-1 interaction domain. We find that hZwint-1–noninteracting mutants still localize to kinetochores. In addition, using fluorescence recovery after photobleaching, we have found that hZW10 residency at metaphase kinetochores is brief (half-time of 13 s). However, during prometaphase or at unattached kinetochores, enhanced green fluorescent protein–hZW10 becomes a stable component of the kinetochore. Moreover, we find that stable hZW10 kinetochore residency at prometaphase kinetochores is dependent on its interaction with hZwint-1, and is essential for mitotic checkpoint arrest.

scholarly journals Switching from a Unicellular to Multicellular Organization in an Aspergillus niger Hypha

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Robert-Jan Bleichrodt ◽  
Marc Hulsman ◽  
Han A. B. Wösten ◽  
Marcel J. T. Reinders
Keyword(s):  
Aspergillus Niger ◽  
Fluorescent Protein ◽  
Stress Conditions ◽  
Physical Contact ◽  
Effective Control ◽  
Mobility Rate ◽  
Content Type ◽  
Green Fluorescent ◽  
First Time ◽  
Compound Concentration

ABSTRACT Pores in fungal septa enable cytoplasmic streaming between hyphae and their compartments. Consequently, the mycelium can be considered unicellular. However, we show here that Woronin bodies close ~50% of the three most apical septa of growing hyphae of Aspergillus niger. The incidence of closure of the 9th and 10th septa was even ≥94%. Intercompartmental streaming of photoactivatable green fluorescent protein (PA-GFP) was not observed when the septa were closed, but open septa acted as a barrier, reducing the mobility rate of PA-GFP ~500 times. This mobility rate decreased with increasing septal age and under stress conditions, likely reflecting a regulatory mechanism affecting septal pore diameter. Modeling revealed that such regulation offers effective control of compound concentration between compartments. Modeling also showed that the incidence of septal closure in A. niger had an even stronger impact on cytoplasmic continuity. Cytoplasm of hyphal compartments was shown not to be in physical contact when separated by more than 4 septa. Together, data show that apical compartments of growing hyphae behave unicellularly, while older compartments have a multicellular organization. IMPORTANCE The hyphae of higher fungi are compartmentalized by porous septa that enable cytosolic streaming. Therefore, it is believed that the mycelium shares cytoplasm. However, it is shown here that the septa of Aspergillus niger are always closed in the oldest part of the hyphae, and therefore, these compartments are physically isolated from each other. In contrast, only part of the septa is closed in the youngest part of the hyphae. Still, compartments in this hyphal part are physically isolated when separated by more than 4 septa. Even open septa act as a barrier for cytoplasmic mixing. The mobility rate through such septa reduces with increasing septal age and under stress conditions. Modeling shows that the septal pore width is set such that its regulation offers maximal control of compound concentration levels within the compartments. Together, we show for the first time that Aspergillus hyphae switch from a unicellular to multicellular organization.

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