scholarly journals Lamin A/C Binding Protein LAP2α Is Required for Nuclear Anchorage of Retinoblastoma Protein

2002 ◽  
Vol 13 (12) ◽  
pp. 4401-4413 ◽  
Author(s):  
Ewa Markiewicz ◽  
Thomas Dechat ◽  
Roland Foisner ◽  
Roy. A Quinlan ◽  
Christopher J. Hutchison
Keyword(s):  
Tissue Culture ◽  
Fluorescent Protein ◽  
Solid Phase ◽  
Retinoblastoma Protein ◽  
Dominant Negative ◽  
Lamin A ◽  
Dependent Manner ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Green Fluorescent

The phosphorylation-dependent anchorage of retinoblastoma protein Rb in the nucleus is essential for its function. We show that its pocket C domain is both necessary and sufficient for nuclear anchorage by transiently expressing green fluorescent protein (GFP) chimeras of Rb fragments in tissue culture cells and by extracting the cells with hypotonic solutions. Solid phase binding assays using glutathioneS-transferase-fusion of Rb pockets A, B, and C revealed a direct association of lamin C exclusively to pocket C. Lamina-associated polypeptide (LAP) 2α, a binding partner of lamins A/C, bound strongly to pocket C and weakly to pocket B. When LAP2α was immunoprecipitated from soluble nuclear fractions, lamins A/C and hypophosphorylated Rb were coprecipitated efficiently. Similarly, immunoprecipitation of expressed GFP-Rb fragments by using anti-GFP antibodies coprecipitated LAP2α, provided that pocket C was present in the GFP chimeras. On redistribution of endogenous lamin A/C and LAP2α into nuclear aggregates by overexpressing dominant negative lamin mutants in tissue culture cells, Rb was also sequestered into these aggregates. In primary skin fibroblasts, LAP2α is expressed in a growth-dependent manner. Anchorage of hypophosphorylated Rb in the nucleus was weakened significantly in the absence of LAP2α. Together, these data suggest that hypophosphorylated Rb is anchored in the nucleus by the interaction of pocket C with LAP2α–lamin A/C complexes.

scholarly journals Examination of Listeria monocytogenes Intracellular Gene Expression by Using the Green Fluorescent Protein ofAequorea victoria

1999 ◽  
Vol 67 (4) ◽  
pp. 1844-1852 ◽  
Author(s):  
Nancy E. Freitag ◽  
Kathleen E. Jacobs
Keyword(s):  
Gene Expression ◽  
Tissue Culture ◽  
Green Fluorescent Protein ◽  
Listeria Monocytogenes ◽  
Host Cell ◽  
Fluorescent Protein ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Green Fluorescent ◽  
Cell Spread

ABSTRACT The ActA protein of Listeria monocytogenes is an essential virulence factor and is required for intracellular bacterial motility and cell-to-cell spread. plcB, cotranscribed withactA, encodes a broad-specificity phospholipase C that contributes to lysis of host cell vacuoles and cell-to-cell spread. Construction of a transcriptional fusion between actA-plcBand the green fluorescent protein gene of Aequorea victoriahas facilitated the detailed examination of patterns ofactA/plcB expression within infected tissue culture cells.actA/plcB expression began approximately 30 min postinfection and was dependent upon entry of L. monocytogenes into the host cytosol. L. monocytogenes Δhly mutants, which are unable to escape from host cell vacuoles, did not express actA/plcB at detectable levels within infected tissue culture cells; however, complementation of thehly defect allowed entry of the bacteria into the host cytoplasm and subsequent actA/plcB expression. These results emphasize the ability of L. monocytogenes to sense the different host cell compartment environments encountered during the course of infection and to regulate virulence gene expression in response.

scholarly journals CRISPR-Cas12a–assisted PCR tagging of mammalian genes

2020 ◽  
Vol 219 (6) ◽  
Author(s):  
Julia Fueller ◽  
Konrad Herbst ◽  
Matthias Meurer ◽  
Krisztina Gubicza ◽  
Bahtiyar Kurtulmus ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Fluorescent Protein ◽  
Mammalian Tissue ◽  
Target Enrichment ◽  
Culture Cells ◽  
Endogenous Expression ◽  
Tissue Culture Cells ◽  
Potential Sources ◽  
Linear Dsdna ◽  
Mammalian Genes

Here we describe a time-efficient strategy for endogenous C-terminal gene tagging in mammalian tissue culture cells. An online platform is used to design two long gene-specific oligonucleotides for PCR with generic template cassettes to create linear dsDNA donors, termed PCR cassettes. PCR cassettes encode the tag (e.g., GFP), a Cas12a CRISPR RNA for cleavage of the target locus, and short homology arms for directed integration via homologous recombination. The integrated tag is coupled to a generic terminator shielding the tagged gene from the co-inserted auxiliary sequences. Co-transfection of PCR cassettes with a Cas12a-encoding plasmid leads to robust endogenous expression of tagged genes, with tagging efficiency of up to 20% without selection, and up to 60% when selection markers are used. We used target-enrichment sequencing to investigate all potential sources of artifacts. Our work outlines a quick strategy particularly suitable for exploratory studies using endogenous expression of fluorescent protein–tagged genes.

Differential Role of PKC Isoforms in GnRH and Phorbol 12-Myristate 13-Acetate Activation of Extracellular Signal-Regulated Kinase and Jun N-Terminal Kinase

Endocrinology ◽  
2010 ◽  
Vol 151 (10) ◽  
pp. 4894-4907 ◽  
Author(s):  
Masha Dobkin-Bekman ◽  
Liat Rahamim Ben-Navi ◽  
Boris Shterntal ◽  
Ludmila Sviridonov ◽  
Fiorenza Przedecki ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Preliminary Evidence ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Pkc Isoforms ◽  
Green Fluorescent ◽  
Jnk Activation ◽  
Erk2 Activation

GnRH is the first key hormone of reproduction. The role of protein kinase C (PKC) isoforms in GnRH-stimulated MAPK [ERK and Jun N-terminal kinase (JNK)] was examined in the αT3-1 and LβT2 gonadotrope cells. Incubation of the cells with GnRH resulted in a protracted activation of ERK1/2 and a slower and more transient activation of JNK1/2. Gonadotropes express conventional PKCα and conventional PKCβII, novel PKCδ, novel PKCε, and novel PKCθ, and atypical PKC-ι/λ. The use of green fluorescent protein-PKC constructs revealed that GnRH induced rapid translocation of PKCα and PKCβII to the plasma membrane, followed by their redistribution to the cytosol. PKCδ and PKCε localized to the cytoplasm and Golgi, followed by the rapid redistribution by GnRH of PKCδ to the perinuclear zone and of PKCε to the plasma membrane. Interestingly, PKCα, PKCβII, and PKCε translocation to the plasma membrane was more pronounced and more prolonged in phorbol-12-myristate-13-acetate (PMA) than in GnRH-treated cells. The use of selective inhibitors and dominant-negative plasmids for the various PKCs has revealed that PKCβII, PKCδ, and PKCε mediate ERK2 activation by GnRH, whereas PKCα, PKCβII, PKCδ, and PKCε mediate ERK2 activation by PMA. Also, PKCα, PKCβII, PKCδ, and PKCε are involved in GnRH and PMA stimulation of JNK1 in a cell-context-dependent manner. We present preliminary evidence that persistent vs. transient redistribution of selected PKCs or redistribution of a given PKC to the perinuclear zone vs. the plasma membrane may dictate its selective role in ERK or JNK activation. Thus, we have described the contribution of selective PKCs to ERK and JNK activation by GnRH.

scholarly journals CRISPR-Cas12a-assisted PCR tagging of mammalian genes

2018 ◽  
Author(s):  
Julia Fueller ◽  
Konrad Herbst ◽  
Matthias Meurer ◽  
Krisztina Gubicza ◽  
Bahtiyar Kurtulmus ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Fluorescent Protein ◽  
Mammalian Tissue ◽  
Target Enrichment ◽  
Culture Cells ◽  
Endogenous Expression ◽  
Tissue Culture Cells ◽  
Potential Sources ◽  
Linear Dsdna ◽  
Mammalian Genes

AbstractHere we describe a time-efficient strategy for endogenous C-terminal gene tagging in mammalian tissue culture cells. An online platform is used to design two long gene-specific oligonucleotides for PCR with generic template cassettes to create linear dsDNA donors, termed PCR cassettes. PCR cassettes encode the tag (e.g. GFP), a Cas12a CRISPR RNA for cleavage of the target locus and short homology arms for directed integration via homologous recombination. The integrated tag is coupled to a generic terminator shielding the tagged gene from the co-inserted auxiliary sequences. Co-transfection of PCR cassettes with a Cas12a-encoding plasmid leads to robust endogenous expression of tagged genes, with tagging efficiency of up to 20% without selection, and up to 60% when selection markers are used. We used target-enrichment sequencing to investigate all potential sources of artefacts. Our work outlines a quick strategy particularly suitable for exploratory studies using endogenous expression of fluorescent protein tagged genes

Cytoplasmic Tubules in Cells Infected with Laryngotracheitis Virus

1969 ◽  
Vol 27 ◽  
pp. 376-377
Author(s):  
A. M. Watrach
Keyword(s):  
Tissue Culture ◽  
Small Proportion ◽  
Chicken Embryo ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Morphologic Characteristics ◽  
Infectious Laryngotracheitis ◽  
Laryngotracheitis Virus ◽  
In Cells

During a study of the development of infectious laryngotracheitis (LT) virus in tissue culture cells, unusual tubular formations were found in the cytoplasm of a small proportion of the affected cells. It is the purpose of this report to describe the morphologic characteristics of the tubules and to discuss their possible association with the development of virus.The source and maintenance of the strain of LT virus have been described. Prior to this study, the virus was passed several times in chicken embryo kidney (CEK) tissue culture cells.

Sulfhydryl bond formation is a prerequisite for proper cycling of centrosomes and chromosomes in mammalian tissue culture cells

1993 ◽  
Vol 51 ◽  
pp. 342-343
Author(s):  
Heide Schatten ◽  
Neidhard Paweletz ◽  
Ron Balczon
Keyword(s):  
Tissue Culture ◽  
Cell Cycle Progression ◽  
Group Formation ◽  
Sulfhydryl Group ◽  
Mammalian Tissue ◽  
Mitotic Chromosomes ◽  
Microtubule Network ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Transmission Electron

To study the role of sulfhydryl group formation during cell cycle progression, mammalian tissue culture cells (PTK2) were exposed to 100¼M 2-mercaptoethanol for 2 to 6 h during their exponential phase of growth. The effects of 2-mercaptoethanol on centrosomes, chromosomes, microtubules, membranes and intermediate filaments were analyzed by transmission electron microscopy (TEM) and by immunofluorescence microscopy (IFM) methods using a human autoimmune antibody directed against centrosomes (SPJ), and a mouse monoclonal antibody directed against tubulin (E7). Chromosomes were affected most by this treatment: premature chromosome condensation was detected in interphase nuclei, and the structure in mitotic chromosomes was altered compared to control cells. This would support previous findings in dividing sea urchin cells in which chromosomes are arrested at metaphase while the centrosome splitting cycle continues. It might also support findings that certairt-sulfhydryl-blocking agents block cyclin destruction. The organization of the microtubule network was scattered probably due to a looser organization of centrosomal material at the interphase centers and at the mitotic poles.

UDP-D-glucose 4-epimerase activity of the tissue culture of white poplars (Populus alba L. var. pyramidalis)

1982 ◽  
Vol 47 (5) ◽  
pp. 1530-1536 ◽  
Author(s):  
Ladislav Bilisics ◽  
Štefan Karácsonyi ◽  
Marta Kubačková
Keyword(s):  
Tissue Culture ◽  
Cell Walls ◽  
Enzyme Preparation ◽  
Uridine Diphosphate ◽  
Populus Alba ◽  
Activity Change ◽  
White Poplar ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Galactose Content

The presence of UDP-D-glucose 4-epimerase (EC 5.1.3.2) in the culture tissue of white poplar was evidenced. As found, the partially purified enzyme preparation contained UDP-D-glucose glucosyltransferase, UDP-D-galactose galactosyltransferase and non-specific enzymes able to cleave the uridine-diphosphate saccharides into the appropriate hexose monophosphates. The activity change of UDP-D-glucose 4-epimerase in tissue culture cells during the growth was in accord with changes in D-galactose content in cell walls and indicated the possibility to regulate the formation of polysaccharides containing D-galactose at the level of production of UDP-D-galactose in cells.

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