scholarly journals A genetically encoded fluorescent biosensor for extracellular l-lactate

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yusuke Nasu ◽  
Ciaran Murphy-Royal ◽  
Yurong Wen ◽  
Jordan N. Haidey ◽  
Rosana S. Molina ◽  
...  
Keyword(s):  
Brain Tissue ◽  
Mammalian Cells ◽  
Waste Product ◽  
Fluorescent Biosensor ◽  
Cellular Resolution ◽  
Resolution Imaging ◽  
Green Fluorescent ◽  
Metabolic Waste Product ◽  
Genetically Encoded Biosensor

Abstractl-Lactate, traditionally considered a metabolic waste product, is increasingly recognized as an important intercellular energy currency in mammals. To enable investigations of the emerging roles of intercellular shuttling of l-lactate, we now report an intensiometric green fluorescent genetically encoded biosensor for extracellular l-lactate. This biosensor, designated eLACCO1.1, enables cellular resolution imaging of extracellular l-lactate in cultured mammalian cells and brain tissue.

scholarly journals A genetically encoded fluorescent biosensor for extracellular L-lactate

2021 ◽  
Author(s):  
Yusuke Nasu ◽  
Ciaran Murphy-Royal ◽  
Yurong Wen ◽  
Jordan Haidey ◽  
M. Rosana S. Molina ◽  
...  
Keyword(s):  
Minimally Invasive ◽  
Brain Tissue ◽  
Mammalian Cells ◽  
Fluorescent Biosensor ◽  
Cellular Resolution ◽  
Resolution Imaging ◽  
Green Fluorescent ◽  
Genetically Encoded Biosensor

AbstractTo enable investigations of the emerging roles of cell-to-cell shuttling of L-lactate, we have developed an intensiometric green fluorescent genetically encoded biosensor for extracellular L-lactate. We demonstrate that this biosensor, designated eLACCO1.1, enables minimally invasive cellular resolution imaging of extracellular L-lactate in cultured mammalian cells and brain tissue.

577 Engineered non-pathogenic synthetic biotic producing L-arginine synergize with PD-1-based cancer immunotherapy

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A611-A611
Author(s):  
Fernando Canale ◽  
Camilla Basso ◽  
Ning Li ◽  
Anna Sokolovska ◽  
Michela Perotti ◽  
...  
Keyword(s):  
T Cells ◽  
Synergistic Effects ◽  
Waste Product ◽  
T Cell Response ◽  
Escherichia Coli Nissle 1917 ◽  
Check Point Inhibitors ◽  
Blocking Antibodies ◽  
Metabolic Waste Product ◽  
Synthetic Biology Approach ◽  
Escherichia Coli Nissle

BackgroundThe availability of L-arginine in tumors is a key determinant of an efficient anti-tumor T cell response. Consequently, the elevation of typically low L-arginine levels within the tumor may greatly potentiate the anti-tumor responses of immune check point inhibitors, such as PD-L1 blocking antibodies. However, currently no means are available to locally increase intra-tumoral L-arginine levels.MethodsWe used a synthetic biology approach to develop an engineered probiotic Escherichia coli Nissle 1917 strain that colonizes tumors and continuously converts ammonia, a metabolic waste product that accumulates in tumors, into L-arginine.ResultsColonization of tumors with these bacteria elevated intra-tumoral L-arginine concentrations, increased the amount of tumor-infiltrating T cells, and had striking synergistic effects with PD-L1 blocking antibodies in the clearance of tumors. The anti-tumor effect of the living therapeutic was mediated by L-arginine and was dependent on T cells.ConclusionsThese results show that engineered microbial therapies enable metabolic modulation of the tumor microenvironment leading to enhanced efficacy of immunotherapies.

scholarly journals Author response: Brain-wide cellular resolution imaging of Cre transgenic zebrafish lines for functional circuit-mapping

2019 ◽  
Author(s):  
Kathryn M Tabor ◽  
Gregory D Marquart ◽  
Christopher Hurt ◽  
Trevor S Smith ◽  
Alexandra K Geoca ◽  
...  
Keyword(s):  
Author Response ◽  
Transgenic Zebrafish ◽  
Cellular Resolution ◽  
Resolution Imaging

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.

scholarly journals A Single Atom Change Facilitates the Membrane Transport of Green Fluorescent Proteins in Mammalian Cells

2019 ◽  
Vol 131 (23) ◽  
pp. 7795-7799 ◽  
Author(s):  
Surendar R. Jakka ◽  
Vijayakumar Govindaraj ◽  
Govindasamy Mugesh
Keyword(s):  
Membrane Transport ◽  
Mammalian Cells ◽  
Fluorescent Proteins ◽  
Single Atom ◽  
Green Fluorescent Proteins ◽  
Green Fluorescent
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