Biochemical Methods to Analyze the Subcellular Localization of NF-κB Proteins Using Cell

2021 ◽  
pp. 19-25
Author(s):  
Davide Vecchiotti ◽  
Daniela Verzella ◽  
Daria Capece ◽  
Mauro Di Vito Nolfi ◽  
Barbara Di Francesco ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Biochemical Methods

Investigating the functional link between TMEM165 and SPCA1

2019 ◽  
Vol 476 (21) ◽  
pp. 3281-3293 ◽  
Author(s):  
Elodie Lebredonchel ◽  
Marine Houdou ◽  
Hans-Heinrich Hoffmann ◽  
Kateryna Kondratska ◽  
Marie-Ange Krzewinski ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Complete Loss ◽  
Protein Family ◽  
Uncharacterized Protein ◽  
Functional Link ◽  
P Type

TMEM165 was highlighted in 2012 as the first member of the Uncharacterized Protein Family 0016 (UPF0016) related to human glycosylation diseases. Defects in TMEM165 are associated with strong Golgi glycosylation abnormalities. Our previous work has shown that TMEM165 rapidly degrades with supraphysiological manganese supplementation. In this paper, we establish a functional link between TMEM165 and SPCA1, the Golgi Ca2+/Mn2+ P-type ATPase pump. A nearly complete loss of TMEM165 was observed in SPCA1-deficient Hap1 cells. We demonstrate that TMEM165 was constitutively degraded in lysosomes in the absence of SPCA1. Complementation studies showed that TMEM165 abundance was directly dependent on SPCA1's function and more specifically its capacity to pump Mn2+ from the cytosol into the Golgi lumen. Among SPCA1 mutants that differentially impair Mn2+ and Ca2+ transport, only the Q747A mutant that favors Mn2+ pumping rescues the abundance and Golgi subcellular localization of TMEM165. Interestingly, the overexpression of SERCA2b also rescues the expression of TMEM165. Finally, this paper highlights that TMEM165 expression is linked to the function of SPCA1.

Subcellular localization of GLUT4 in nonstimulated and insulin-stimulated soleus muscle of rat

Diabetes ◽  
1992 ◽  
Vol 41 (2) ◽  
pp. 215-221 ◽  
Author(s):  
A. Bornemann ◽  
T. Ploug ◽  
H. Schmalbruch
Keyword(s):  
Subcellular Localization ◽  
Soleus Muscle

Single-Molecule Imaging of Active Mitochondrial Nitroreductases using a Photo-Crosslinking Fluorescent Sensor

2019 ◽  
Author(s):  
Zacharias Thiel ◽  
Pablo Rivera-Fuentes
Keyword(s):  
Subcellular Localization ◽  
Fluorescent Probe ◽  
Single Molecule ◽  
Mammalian Cells ◽  
Fluorescent Sensor ◽  
Biological Functions ◽  
Localization Microscopy ◽  
Drug Activation ◽  
Nitroreductase Activity ◽  
Single Molecule Localization Microscopy

Many biomacromolecules are known to cluster in microdomains with specific subcellular localization. In the case of enzymes, this clustering greatly defines their biological functions. Nitroreductases are enzymes capable of reducing nitro groups to amines and play a role in detoxification and pro-drug activation. Although nitroreductase activity has been detected in mammalian cells, the subcellular localization of this activity remains incompletely characterized. Here, we report a fluorescent probe that enables super-resolved imaging of pools of nitroreductase activity within mitochondria. This probe is activated sequentially by nitroreductases and light to give a photo-crosslinked adduct of active enzymes. In combination with a general photoactivatable marker of mitochondria, we performed two-color, threedimensional, single-molecule localization microscopy. These experiments allowed us to image the sub-mitochondrial organization of microdomains of nitroreductase activity.<br>

Single-Molecule Imaging of Active Mitochondrial Nitroreductases using a Photo-Crosslinking Fluorescent Sensor

2019 ◽  
Author(s):  
Zacharias Thiel ◽  
Pablo Rivera-Fuentes
Keyword(s):  
Subcellular Localization ◽  
Fluorescent Probe ◽  
Single Molecule ◽  
Mammalian Cells ◽  
Fluorescent Sensor ◽  
Biological Functions ◽  
Localization Microscopy ◽  
Drug Activation ◽  
Nitroreductase Activity ◽  
Single Molecule Localization Microscopy

Many biomacromolecules are known to cluster in microdomains with specific subcellular localization. In the case of enzymes, this clustering greatly defines their biological functions. Nitroreductases are enzymes capable of reducing nitro groups to amines and play a role in detoxification and pro-drug activation. Although nitroreductase activity has been detected in mammalian cells, the subcellular localization of this activity remains incompletely characterized. Here, we report a fluorescent probe that enables super-resolved imaging of pools of nitroreductase activity within mitochondria. This probe is activated sequentially by nitroreductases and light to give a photo-crosslinked adduct of active enzymes. In combination with a general photoactivatable marker of mitochondria, we performed two-color, threedimensional, single-molecule localization microscopy. These experiments allowed us to image the sub-mitochondrial organization of microdomains of nitroreductase activity.<br>

An efficient transient assays system using Agrobacterium-mediated transformation of onion (Allium cepa) epidermal cells

2020 ◽  
Vol 80 (03) ◽  
Author(s):  
Yu-Miao Zhang ◽  
Jun Wang ◽  
Tao Wu
Keyword(s):  
Subcellular Localization ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Epidermal Cells ◽  
Cyclin Dependent Kinase ◽  
Protein Subcellular Localization ◽  
Protein Protein Interactions ◽  
Efficient System ◽  
Protein Protein Interaction ◽  
Onion Epidermal Cells

In this study, the Agrobacterium infection medium, infection duration, detergent, and cell density were optimized. The sorghum-based infection medium (SbIM), 10-20 min infection time, addition of 0.01% Silwet L-77, and Agrobacterium optical density at 600 nm (OD600), improved the competence of onion epidermal cells to support Agrobacterium infection at >90% efficiency. Cyclin-dependent kinase D-2 (CDKD-2) and cytochrome c-type biogenesis protein (CYCH), protein-protein interactions were localized. The optimized procedure is a quick and efficient system for examining protein subcellular localization and protein-protein interaction.

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