scholarly journals Prophase Microtubule Arrays Undergo Flux-like Behavior in Mammalian Cells

2007 ◽  
Vol 18 (10) ◽  
pp. 3993-4002 ◽  
Author(s):  
Nick P. Ferenz ◽  
Patricia Wadsworth
Keyword(s):  
Mammalian Cells ◽  
Marked Reduction ◽  
Fluorescent Protein ◽  
Model Systems ◽  
Microtubule Organization ◽  
Spindle Microtubule ◽  
Nuclear Envelope Breakdown ◽  
Wide Range ◽  
Green Fluorescent ◽  
Poleward Flux

In higher eukaryotic cells, microtubules within metaphase and anaphase spindles undergo poleward flux, the slow, poleward movement of tubulin subunits through the spindle microtubule lattice. Although a number of studies have documented this phenomenon across a wide range of model systems, the possibility of poleward flux before nuclear envelope breakdown (NEB) has not been examined. Using a mammalian cell line expressing photoactivatable green fluorescent protein (GFP)-tubulin, we observe microtubule motion, both toward and away from centrosomes, at a wide range of rates (0.5–4.5 μm/min) in prophase cells. Rapid microtubule motion in both directions is dynein dependent. In contrast, slow microtubule motion, which occurs at rates consistent with metaphase flux, is insensitive to inhibition of dynein but sensitive to perturbation of Eg5 and Kif2a, two proteins with previously documented roles in flux. Our results demonstrate that microtubules in prophase cells are unexpectedly dynamic and that a subpopulation of these microtubules shows motion that is consistent with flux. We propose that the marked reduction in rate and directionality of microtubule motion from prophase to metaphase results from changes in microtubule organization during spindle formation.

scholarly journals Dual Targeting of Osh1p, a Yeast Homologue of Oxysterol-binding Protein, to both the Golgi and the Nucleus-Vacuole Junction

2001 ◽  
Vol 12 (6) ◽  
pp. 1633-1644 ◽  
Author(s):  
Timothy P. Levine ◽  
Sean Munro
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Cholesterol Homeostasis ◽  
Deletion Mapping ◽  
Ph Domain ◽  
Oxysterol Binding Protein ◽  
Wide Range ◽  
Ph Domains ◽  
Green Fluorescent

Oxysterol binding protein (OSBP) is the only protein known to bind specifically to the group of oxysterols with potent effects on cholesterol homeostasis. Although the function of OSBP is currently unknown, an important role is implicated by the existence of multiple homologues in all eukaryotes so far examined. OSBP and a subset of homologues contain pleckstrin homology (PH) domains. Such domains are responsible for the targeting of a wide range of proteins to the plasma membrane. In contrast, OSBP is a peripheral protein of Golgi membranes, and its PH domain targets to the trans-Golgi network of mammalian cells. In this article, we have characterized Osh1p, Osh2p, and Osh3p, the three homologues of OSBP inSaccharomyces cerevisiae that contain PH domains. Examination of a green fluorescent protein (GFP) fusion to Osh1p revealed a striking dual localization with the protein present on both the late Golgi, and in the recently described nucleus-vacuole (NV) junction. Deletion mapping revealed that the PH domain of Osh1p specified targeting to the late Golgi, and an ankyrin repeat domain targeting to the NV junction, the first such targeting domain identified for this structure. GFP fusions to Osh2p and Osh3p showed intracellular distributions distinct from that of Osh1p, and their PH domains appear to contribute to their differing localizations.

scholarly journals Rtn1p Is Involved in Structuring the Cortical Endoplasmic Reticulum

2006 ◽  
Vol 17 (7) ◽  
pp. 3009-3020 ◽  
Author(s):  
Johan-Owen De Craene ◽  
Jeff Coleman ◽  
Paula Estrada de Martin ◽  
Marc Pypaert ◽  
Scott Anderson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Cell Cortex ◽  
Proteomic Screen ◽  
Wide Range ◽  
Membrane Spanning ◽  
Green Fluorescent ◽  
Hydrophilic Loop ◽  
Yeast Mutants

The endoplasmic reticulum (ER) contains both cisternal and reticular elements in one contiguous structure. We identified rtn1Δ in a systematic screen for yeast mutants with altered ER morphology. The ER in rtn1Δ cells is predominantly cisternal rather than reticular, yet the net surface area of ER is not significantly changed. Rtn1-green fluorescent protein (GFP) associates with the reticular ER at the cell cortex and with the tubules that connect the cortical ER to the nuclear envelope, but not with the nuclear envelope itself. Rtn1p overexpression also results in an altered ER structure. Rtn proteins are found on the ER in a wide range of eukaryotes and are defined by two membrane-spanning domains flanking a conserved hydrophilic loop. Our results suggest that Rtn proteins may direct the formation of reticulated ER. We independently identified Rtn1p in a proteomic screen for proteins associated with the exocyst vesicle tethering complex. The conserved hydophilic loop of Rtn1p binds to the exocyst subunit Sec6p. Overexpression of this loop results in a modest accumulation of secretory vesicles, suggesting impaired exocyst function. The interaction of Rtn1p with the exocyst at the bud tip may trigger the formation of a cortical ER network in yeast buds.

scholarly journals Vibrio cholerae Triggers SOS and Mutagenesis in Response to a Wide Range of Antibiotics: a Route towards Multiresistance

2011 ◽  
Vol 55 (5) ◽  
pp. 2438-2441 ◽  
Author(s):  
Zeynep Baharoglu ◽  
Didier Mazel
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Vibrio Cholerae ◽  
Fluorescent Protein ◽  
Resistance Development ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Green Fluorescent ◽  
Regulated Promoters

ABSTRACTAntibiotic resistance development has been linked to the bacterial SOS stress response. InEscherichia coli, fluoroquinolones are known to induce SOS, whereas other antibiotics, such as aminoglycosides, tetracycline, and chloramphenicol, do not. Here we address whether various antibiotics induce SOS inVibrio cholerae. Reporter green fluorescent protein (GFP) fusions were used to measure the response of SOS-regulated promoters to subinhibitory concentrations of antibiotics. We show that unlike the situation withE. coli, all these antibiotics induce SOS inV. cholerae.

scholarly journals New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters

2016 ◽  
Vol 82 (8) ◽  
pp. 2240-2246 ◽  
Author(s):  
Alex I. Kanno ◽  
Cibelly Goulart ◽  
Henrique K. Rofatto ◽  
Sergio C. Oliveira ◽  
Luciana C. C. Leite ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Expression Vectors ◽  
Content Type ◽  
Expression Levels ◽  
Wide Range ◽  
Mycobacteriophage L5 ◽  
Green Fluorescent ◽  
Selection Of

ABSTRACTThe expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such asMycobacterium bovisBCG orM. smegmatiswas made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinantM. smegmatisbacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in bothM. smegmatisandM. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of theSchistosoma mansoniantigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response.

scholarly journals Characterization of Cep135, a novel coiled-coil centrosomal protein involved in microtubule organization in mammalian cells

2002 ◽  
Vol 156 (1) ◽  
pp. 87-100 ◽  
Author(s):  
Toshiro Ohta ◽  
Russell Essner ◽  
Jung-Hwa Ryu ◽  
Robert E. Palazzo ◽  
Yumi Uetake ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Coiled Coil ◽  
Amino Acid Residues ◽  
Protein Overexpression ◽  
Microtubule Organization ◽  
Microtubule Network ◽  
Centrosomal Protein ◽  
Wide Range ◽  
Spindle Microtubules

By using monoclonal antibodies raised against isolated clam centrosomes, we have identified a novel 135-kD centrosomal protein (Cep135), present in a wide range of organisms. Cep135 is located at the centrosome throughout the cell cycle, and localization is independent of the microtubule network. It distributes throughout the centrosomal area in association with the electron-dense material surrounding centrioles. Sequence analysis of cDNA isolated from CHO cells predicted a protein of 1,145–amino acid residues with extensive α-helical domains. Expression of a series of deletion constructs revealed the presence of three independent centrosome-targeting domains. Overexpression of Cep135 resulted in the accumulation of unique whorl-like particles in both the centrosome and the cytoplasm. Although their size, shape, and number varied according to the level of protein expression, these whorls were composed of parallel dense lines arranged in a 6-nm space. Altered levels of Cep135 by protein overexpression and/or suppression of endogenous Cep135 by RNA interference caused disorganization of interphase and mitotic spindle microtubules. Thus, Cep135 may play an important role in the centrosomal function of organizing microtubules in mammalian cells.

scholarly journals Dynamin-related Protein Drp1 Is Required for Mitochondrial Division in Mammalian Cells

2001 ◽  
Vol 12 (8) ◽  
pp. 2245-2256 ◽  
Author(s):  
Elena Smirnova ◽  
Lorena Griparic ◽  
Dixie-Lee Shurland ◽  
Alexander M. van der Bliek
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Subcellular Fractionation ◽  
Related Protein ◽  
Direct Role ◽  
Mitochondrial Division ◽  
Transfected Cells ◽  
Green Fluorescent ◽  
Time Lapse Photography

Mutations in the human dynamin-related protein Drp1 cause mitochondria to form perinuclear clusters. We show here that these mitochondrial clusters consist of highly interconnected mitochondrial tubules. The increased connectivity between mitochondria indicates that the balance between mitochondrial division and fusion is shifted toward fusion. Such a shift is consistent with a block in mitochondrial division. Immunofluorescence and subcellular fractionation show that endogenous Drp1 is localized to mitochondria, which is also consistent with a role in mitochondrial division. A direct role in mitochondrial division is suggested by time-lapse photography of transfected cells, in which green fluorescent protein fused to Drp1 is concentrated in spots that mark actual mitochondrial division events. We find that purified human Drp1 can self-assemble into multimeric ring-like structures with dimensions similar to those of dynamin multimers. The structural and functional similarities between dynamin and Drp1 suggest that Drp1 wraps around the constriction points of dividing mitochondria, analogous to dynamin collars at the necks of budding vesicles. We conclude that Drp1 contributes to mitochondrial division in mammalian cells.

Quantitative measurement of mammalian chromosome mitotic loss rates using the green fluorescent protein

1999 ◽  
Vol 112 (16) ◽  
pp. 2705-2714
Author(s):  
E.M. Burns ◽  
L. Christopoulou ◽  
P. Corish ◽  
C. Tyler-Smith
Keyword(s):  
Green Fluorescent Protein ◽  
Mammalian Cells ◽  
Quantitative Measurement ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Chromosome Loss ◽  
Facs Analysis ◽  
Fluorescent Cells ◽  
Green Fluorescent ◽  
Loss Rates

We have measured the mitotic loss rates of mammalian chromosomes in cultured cells. The green fluorescent protein (GFP) gene was incorporated into a non-essential chromosome so that cells containing the chromosome fluoresced green, while those lacking it did not. The proportions of fluorescent and non-fluorescent cells were measured by fluorescence activated cell sorter (FACS) analysis. Loss rates ranged from 0.005% to 0.20% per cell division in mouse LA-9 cells, and from 0.02% to 0.40% in human HeLa cells. The rate of loss was elevated by treatment with aneugens, demonstrating that the system rapidly identifies agents which induce chromosome loss in mammalian cells.

scholarly journals Visualization of the Peroxisomal Compartment in Living Mammalian Cells: Dynamic Behavior and Association with Microtubules

1997 ◽  
Vol 136 (1) ◽  
pp. 71-80 ◽  
Author(s):  
Erik A.C. Wiemer ◽  
Thibaut Wenzel ◽  
Thomas J. Deerinck ◽  
Mark H. Ellisman ◽  
Suresh Subramani
Keyword(s):  
Mammalian Cells ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Small Subset ◽  
Subcellular Compartment ◽  
Directional Movement ◽  
Green Fluorescent

Peroxisomes in living CV1 cells were visualized by targeting the green fluorescent protein (GFP) to this subcellular compartment through the addition of a COOH-terminal peroxisomal targeting signal 1 (GFP–PTS1). The organelle dynamics were examined and analyzed using time-lapse confocal laser scanning microscopy. Two types of movement could be distinguished: a relatively slow, random, vibration-like movement displayed by the majority (∼95%) of the peroxisomes, and a saltatory, fast directional movement displayed by a small subset (∼5%) of the peroxisomes. In the latter instance, peak velocities up to 0.75 μm/s and sustained directional velocities up to 0.45 μm/s over 11.5 μm were recorded. Only the directional type of motion appeared to be energy dependent, whereas the vibrational movement continued even after the cells were depleted of energy. Treatment of cells, transiently expressing GFP–PTS1, with microtubule-destabilizing agents such as nocodazole, vinblastine, and demecolcine clearly altered peroxisome morphology and subcellular distribution and blocked the directional movement. In contrast, the microtubule-stabilizing compound paclitaxel, or the microfilament-destabilizing drugs cytochalasin B or D, did not exert these effects. High resolution confocal analysis of cells expressing GFP–PTS1 and stained with anti-tubulin antibodies revealed that many peroxisomes were associated with microtubules. The GFP–PTS1–labeled peroxisomes were found to distribute themselves in a stochastic, rather than ordered, manner to daughter cells at the time of mitosis.

Supercharged green fluorescent protein delivers HPV16E7 DNA and protein into mammalian cells in vitro and in vivo

2018 ◽  
Vol 194 ◽  
pp. 29-39 ◽  
Author(s):  
Fatemeh Motevalli ◽  
Azam Bolhassani ◽  
Shilan Hesami ◽  
Sepideh Shahbazi
Keyword(s):  
Green Fluorescent Protein ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Green Fluorescent
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