scholarly journals A new live-cell reporter strategy to simultaneously monitor mitochondrial biogenesis and morphology

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Linn Iren Hodneland Nilsson ◽  
Ina Katrine Nitschke Pettersen ◽  
Julie Nikolaisen ◽  
David Micklem ◽  
Hege Avsnes Dale ◽  
...  
Keyword(s):  
Mitochondrial Biogenesis ◽  
Fluorescent Protein ◽  
Live Cell ◽  
Mitochondrial Morphology ◽  
Reporter Construct ◽  
Nuclear Respiratory Factor ◽  
Nuclear Respiratory Factor 1 ◽  
Vital Functions ◽  
Dependent Protein ◽  
Green Fluorescent

Abstract Changes in mitochondrial amount and shape are intimately linked to maintenance of cell homeostasis via adaptation of vital functions. Here, we developed a new live-cell reporter strategy to simultaneously monitor mitochondrial biogenesis and morphology. This was achieved by making a genetic reporter construct where a master regulator of mitochondrial biogenesis, nuclear respiratory factor 1 (NRF-1), controls expression of mitochondria targeted green fluorescent protein (mitoGFP). HeLa cells with the reporter construct demonstrated inducible expression of mitoGFP upon activation of AMP-dependent protein kinase (AMPK) with AICAR. We established stable reporter cells where the mitoGFP reporter activity corresponded with mitochondrial biogenesis both in magnitude and kinetics, as confirmed by biochemical markers and confocal microscopy. Quantitative 3D image analysis confirmed accordant increase in mitochondrial biomass, in addition to filament/network promoting and protecting effects on mitochondrial morphology, after treatment with AICAR. The level of mitoGFP reversed upon removal of AICAR, in parallel with decrease in mtDNA. In summary, we here present a new GFP-based genetic reporter strategy to study mitochondrial regulation and dynamics in living cells. This combinatorial reporter concept can readily be transferred to other cell models and contexts to address specific physiological mechanisms.

scholarly journals Red-Shifted Aminated Derivatives of GFP Chromophore for Live-Cell Protein Labeling with Lipocalins

2018 ◽  
Vol 19 (12) ◽  
pp. 3778 ◽  
Author(s):  
Nina Bozhanova ◽  
Mikhail Baranov ◽  
Nadezhda Baleeva ◽  
Alexey Gavrikov ◽  
Alexander Mishin
Keyword(s):  
Green Fluorescent Protein ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Live Cell ◽  
Protein Labeling ◽  
Cell Protein ◽  
Gfp Chromophore ◽  
Green Fluorescent ◽  
Derivatives Of

Fluorogens are an attractive type of dye for imaging applications, eliminating time-consuming washout steps from staining protocols. With just a handful of reported fluorogen-protein pairs, mostly in the green region of spectra, there is a need for the expansion of their spectral range. Still, the origins of solvatochromic and fluorogenic properties of the chromophores suitable for live-cell imaging are poorly understood. Here we report on the synthesis and labeling applications of novel red-shifted fluorogenic cell-permeable green fluorescent protein (GFP) chromophore analogs.

scholarly journals Live-Cell Analysis of a Green Fluorescent Protein-Tagged Herpes Simplex Virus Infection

1999 ◽  
Vol 73 (5) ◽  
pp. 4110-4119 ◽  
Author(s):  
Gillian Elliott ◽  
Peter O’Hare
Keyword(s):  
Herpes Simplex Virus ◽  
Green Fluorescent Protein ◽  
Herpes Simplex ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Live Cell ◽  
Cell Periphery ◽  
Green Fluorescent ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Many stages of the herpes simplex virus maturation pathway have not yet been defined. In particular, little is known about the assembly of the virion tegument compartment and its subsequent incorporation into maturing virus particles. Here we describe the construction of a herpes simplex virus type 1 (HSV-1) recombinant in which we have replaced the gene encoding a major tegument protein, VP22, with a gene expressing a green fluorescent protein (GFP)-VP22 fusion protein (GFP-22). We show that this virus has growth properties identical to those of the parental virus and that newly synthesized GFP-22 is detectable in live cells as early as 3 h postinfection. Moreover, we show that GFP-22 is incorporated into the HSV-1 virion as efficiently as VP22, resulting in particles which are visible by fluorescence microscopy. Consequently, we have used time lapse confocal microscopy to monitor GFP-22 in live-cell infection, and we present time lapse animations of GFP-22 localization throughout the virus life cycle. These animations demonstrate that GFP-22 is present in a diffuse cytoplasmic location when it is initially expressed but evolves into particulate material which travels through an exclusively cytoplasmic pathway to the cell periphery. In this way, we have for the first time visualized the trafficking of a herpesvirus structural component within live, infected cells.

scholarly journals A Mutation in a Novel Yeast Proteasomal Gene,RPN11/MPR1, Produces a Cell Cycle Arrest, Overreplication of Nuclear and Mitochondrial DNA, and an Altered Mitochondrial Morphology

1998 ◽  
Vol 9 (10) ◽  
pp. 2917-2931 ◽  
Author(s):  
Teresa Rinaldi ◽  
Carlo Ricci ◽  
Danilo Porro ◽  
Monique Bolotin-Fukuhara ◽  
Laura Frontali
Keyword(s):  
Mitochondrial Dna ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Mitochondrial Morphology ◽  
Nonpermissive Temperature ◽  
Protein Fusion ◽  
In The Wild ◽  
A Cell ◽  
Green Fluorescent ◽  
Cytoplasmic Structures

We report here the functional characterization of an essentialSaccharomyces cerevisiae gene, MPR1, coding for a regulatory proteasomal subunit for which the name Rpn11p has been proposed. For this study we made use of thempr1-1 mutation that causes the following pleiotropic defects. At 24°C growth is delayed on glucose and impaired on glycerol, whereas no growth is seen at 36°C on either carbon source. Microscopic observation of cells growing on glucose at 24°C shows that most of them bear a large bud, whereas mitochondrial morphology is profoundly altered. A shift to the nonpermissive temperature produces aberrant elongated cell morphologies, whereas the nucleus fails to divide. Flow cytometry profiles after the shift to the nonpermissive temperature indicate overreplication of both nuclear and mitochondrial DNA. Consistently with the identification of Mpr1p with a proteasomal subunit, the mutation is complemented by the human POH1proteasomal gene. Moreover, the mpr1-1 mutant grown to stationary phase accumulates ubiquitinated proteins. Localization of the Rpn11p/Mpr1p protein has been studied by green fluorescent protein fusion, and the fusion protein has been found to be mainly associated to cytoplasmic structures. For the first time, a proteasomal mutation has also revealed an associated mitochondrial phenotype. We actually showed, by the use of [rho°] cells derived from the mutant, that the increase in DNA content per cell is due in part to an increase in the amount of mitochondrial DNA. Moreover, microscopy of mpr1-1 cells grown on glucose showed that multiple punctate mitochondrial structures were present in place of the tubular network found in the wild-type strain. These data strongly suggest that mpr1-1 is a valuable tool with which to study the possible roles of proteasomal function in mitochondrial biogenesis.

scholarly journals KlRHO1 and KlPKC1 are essential for cell integrity signalling in Kluyveromyces lactis

Microbiology ◽  
2006 ◽  
Vol 152 (9) ◽  
pp. 2635-2649 ◽  
Author(s):  
Rosaura Rodicio ◽  
Sabrina Koch ◽  
Hans-Peter Schmitz ◽  
Jürgen J. Heinisch
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Kluyveromyces Lactis ◽  
Small Gtpase ◽  
Cell Integrity ◽  
Kinase C ◽  
Vital Functions ◽  
Rigid Cell Wall ◽  
Division Process ◽  
Green Fluorescent

Cell integrity in yeasts is ensured by a rigid cell wall whose synthesis is triggered by a MAP kinase-mediated signal-transduction cascade. Upstream regulatory components of this pathway in Saccharomyces cerevisiae involve a single protein kinase C, which is regulated by interaction with the small GTPase Rho1. Here, two genes were isolated which encode these proteins from Kluyveromyces lactis (KlPKC1 and KlRHO1). Sequencing showed ORFs which encode proteins of 1161 and 208 amino acids, respectively. The deduced proteins shared 59 and 85 % overall amino acid identities, respectively, with their homologues from S. cerevisiae. Null mutants in both genes were non-viable, as shown by tetrad analyses of the heterozygous diploid strains. Overexpression of the KlRHO1 gene under the control of the ScGAL1 promoter severely impaired growth in both S. cerevisiae and K. lactis. On the other hand, a similar construct with KlPKC1 did not show a pronounced phenotype. Two-hybrid analyses showed interaction between Rho1 and Pkc1 for the K. lactis proteins and their S. cerevisiae homologues. A green fluorescent protein (GFP) fusion to the C-terminal end of KlPkc1 located the protein to patches in the growing bud, and at certain stages of the division process also to the bud neck. N-terminal GFP fusions to KlRho1 localized mainly to the cell surface (presumably the cytoplasmic side of the plasma membrane) and to the vacuole, with some indications of traffic from the former to the latter. Thus, KlPkc1 and KlRho1 have been shown to serve vital functions in K. lactis, to interact in cell integrity signalling and to traffic between the plasma membrane and the vacuole.

Phosphodiesterase-4 gates the ability of protein kinase A to phosphorylate G-protein receptor kinase-2 and influence its translocation

2006 ◽  
Vol 34 (4) ◽  
pp. 474-475 ◽  
Author(s):  
M.D. Houslay ◽  
G.S. Baillie
Keyword(s):  
Protein Kinase ◽  
G Protein ◽  
Fluorescent Protein ◽  
Resting Cells ◽  
Receptor Kinase ◽  
Human Embryonic Kidney Cells ◽  
Phosphodiesterase 4 ◽  
Protein Receptor ◽  
Dependent Protein ◽  
Green Fluorescent

Challenge of the β2Ar (β2-adrenergic receptor) with isoprenaline in HEK-293β2 cells (human embryonic kidney cells stably overexpressing a FLAG- and green fluorescent protein-tagged β2Ar) results in the PKA (cAMP-dependent protein kinase) phosphorylation of GRK2 (G-protein receptor kinase-2). This response was enhanced when PDE4 (phosphodiesterase-4) activity was attenuated using either rolipram, a PDE4-selective inhibitor, or with siRNA (small interfering RNA) knockdown of both PDE4B and PDE4D. Rolipram also facilitated GRK2 recruitment to the membrane and phosphorylation of the β2Ar by GRK2 in response to isoprenaline challenge of cells. In resting cells, rolipram treatment alone is sufficient to promote PKA phosphorylation of GRK2, with consequential effects on GRK2 translocation and GRK2 phosphorylation of the β2Ar. Similar effects are observed in cardiac myocytes. We propose that PDE4 activity protects GRK2 from inappropriate phosphorylation by PKA in resting cells that might have occurred through fluctuations in basal cAMP levels. Thus PDE4 gates the action of PKA to phosphorylate GRK2.

scholarly journals Quantitation of mitochondrial dynamics by photolabeling of individual organelles shows that mitochondrial fusion is blocked during the Bax activation phase of apoptosis

2004 ◽  
Vol 164 (4) ◽  
pp. 493-499 ◽  
Author(s):  
Mariusz Karbowski ◽  
Damien Arnoult ◽  
Hsiuchen Chen ◽  
David C. Chan ◽  
Carolyn L. Smith ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Mitochondrial Dynamics ◽  
Dynamic Balance ◽  
Mitochondrial Fusion ◽  
Confocal Imaging ◽  
Time Range ◽  
Mitochondrial Morphology ◽  
Green Fluorescent ◽  
Mitochondrial Membrane Permeabilization ◽  
Activation Phase

A dynamic balance of organelle fusion and fission regulates mitochondrial morphology. During apoptosis this balance is altered, leading to an extensive fragmentation of the mitochondria. Here, we describe a novel assay of mitochondrial dynamics based on confocal imaging of cells expressing a mitochondrial matrix–targeted photoactivable green fluorescent protein that enables detection and quantification of organelle fusion in living cells. Using this assay, we visualize and quantitate mitochondrial fusion rates in healthy and apoptotic cells. During apoptosis, mitochondrial fusion is blocked independently of caspase activation. The block in mitochondrial fusion occurs within the same time range as Bax coalescence on the mitochondria and outer mitochondrial membrane permeabilization, and it may be a consequence of Bax/Bak activation during apoptosis.

Beneficial Effect of Flavone Derivatives on Aβ-Induced Memory Deficit Is Mediated by Peroxisome Proliferator-Activated Receptor γ Coactivator 1α

2015 ◽  
Vol 34 (3) ◽  
pp. 274-283 ◽  
Author(s):  
Farshad Arsalandeh ◽  
Shahin Ahmadian ◽  
Forough Foolad ◽  
Fariba Khodagholi ◽  
Mahdi M. Farimani ◽  
...  
Keyword(s):  
Mitochondrial Biogenesis ◽  
Neuroprotective Effect ◽  
Memory Function ◽  
Adenosine Monophosphate ◽  
Peroxisome Proliferator ◽  
Protective Effects ◽  
Peroxisome Proliferator Activated Receptor ◽  
Nuclear Respiratory Factor ◽  
Mitochondrial Transcription Factor ◽  
Nuclear Respiratory Factor 1

In the present study, the neuroprotective effect of 5-hydroxy-6,7,4′-trimethoxyflavone (flavone 1), a natural flavone, was investigated in comparison with another flavone, 5,7,4′-trihydroxyflavone (flavone 2) on the hippocampus of amyloid beta (Aβ)-injected rats. Rats were treated with the 2 flavones (1 mg/kg/d) for 1 week before Aβ injection. Seven days after Aβ administration, memory function of rats was assessed in a passive avoidance test (PAT). Changes in the levels of mitochondrial transcription factor A (TFAM), peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α), phospho-adenosine monophosphate (AMP)-activated protein kinase (pAMPK), AMPK, phospho-cAMP-responsive element-binding protein (CREB), CREB, and nuclear respiratory factor 1 (NRF-1) proteins were determined by Western blot analysis. Our results showed an improvement in memory in rats pretreated with flavonoids. At the molecular level, phosphorylation of CREB, known as the master modulator of memory processes, increased. On the other hand, the level of mitochondrial biogenesis factors, PGC-1α and its downstream molecules NRF-1 and TFAM significantly increased by dietary administration of 2 flavones. In addition, flavone 1 and flavone 2 prevented mitochondrial swelling and mitochondrial membrane potential reduction. Our results provided evidence that flavone 1 is more effective than flavone 2 presumably due to its O-methylated groups. In conclusion, it seems that in addition to classical antioxidant effect, flavones exert part of their protective effects through mitochondrial biogenesis.

scholarly journals 3P306 Monitoring of mitochondrial morphology in. a single living cell using green fluorescent protein(Bioimaging,Poster Presentations)

2007 ◽  
Vol 47 (supplement) ◽  
pp. S279
Author(s):  
Toshifumi Takayama ◽  
Michihiko Sato ◽  
Fumihiko Fujii ◽  
Masataka Kinjo ◽  
Zhonggang Feng ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Living Cell ◽  
Fluorescent Protein ◽  
Mitochondrial Morphology ◽  
Single Living Cell ◽  
Green Fluorescent ◽  
Poster Presentations ◽  
Single Living

scholarly journals Impact of signaling microcompartment geometry on GPCR dynamics in live retinal photoreceptors

2012 ◽  
Vol 140 (3) ◽  
pp. 249-266 ◽  
Author(s):  
Mehdi Najafi ◽  
Mohammad Haeri ◽  
Barry E. Knox ◽  
William E. Schiesser ◽  
Peter D. Calvert
Keyword(s):  
Green Fluorescent Protein ◽  
Molecular Diffusion ◽  
Fluorescent Protein ◽  
Lateral Diffusion ◽  
Live Cell ◽  
Confocal Imaging ◽  
Slow Phase ◽  
Protein Diffusion ◽  
Green Fluorescent ◽  
The Impact

G protein–coupled receptor (GPCR) cascades rely on membrane protein diffusion for signaling and are generally found in spatially constrained subcellular microcompartments. How the geometry of these microcompartments impacts cascade activities, however, is not understood, primarily because of the inability of current live cell–imaging technologies to resolve these small structures. Here, we examine the dynamics of the GPCR rhodopsin within discrete signaling microcompartments of live photoreceptors using a novel high resolution approach. Rhodopsin fused to green fluorescent protein variants, either enhanced green fluorescent protein (EGFP) or the photoactivatable PAGFP (Rho-E/PAGFP), was expressed transgenically in Xenopus laevis rod photoreceptors, and the geometries of light signaling microcompartments formed by lamellar disc membranes and their incisure clefts were resolved by confocal imaging. Multiphoton fluorescence relaxation after photoconversion experiments were then performed with a Ti–sapphire laser focused to the diffraction limit, which produced small sub–cubic micrometer volumes of photoconverted molecules within the discrete microcompartments. A model of molecular diffusion was developed that allows the geometry of the particular compartment being examined to be specified. This was used to interpret the experimental results. Using this unique approach, we showed that rhodopsin mobility across the disc surface was highly heterogeneous. The overall relaxation of Rho-PAGFP fluorescence photoactivated within a microcompartment was biphasic, with a fast phase lasting several seconds and a slow phase of variable duration that required up to several minutes to reach equilibrium. Local Rho-EGFP diffusion within defined compartments was monotonic, however, with an effective lateral diffusion coefficient Dlat = 0.130 ± 0.012 µm2s−1. Comparison of rhodopsin-PAGFP relaxation time courses with model predictions revealed that microcompartment geometry alone may explain both fast local rhodopsin diffusion and its slow equilibration across the greater disc membrane. Our approach has for the first time allowed direct examination of GPCR dynamics within a live cell signaling microcompartment and a quantitative assessment of the impact of compartment geometry on GPCR activity.

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