Cellular Signaling and Protein−Protein Interactions Studied Using Fluorescence Recovery after Photobleaching†

2004 ◽  
Vol 108 (29) ◽  
pp. 10540-10546 ◽  
Author(s):  
Star M. Dunham ◽  
Haridas E. Pudavar ◽  
Paras N. Prasad ◽  
Michal K. Stachowiak
Keyword(s):  
Protein Interactions ◽  
Fluorescence Recovery After Photobleaching ◽  
Cellular Signaling ◽  
Fluorescence Recovery ◽  
Protein Protein Interactions

scholarly journals Microfluidic immobilization and subcellular imaging of developing Caenorhabditis elegans

2017 ◽  
Author(s):  
Jordan Shivers ◽  
Sravanti Uppaluri ◽  
Clifford P. Brangwynne
Keyword(s):  
Larval Development ◽  
Protein Interactions ◽  
3D Imaging ◽  
Fluorescence Recovery After Photobleaching ◽  
Model Organism ◽  
Fluorescence Recovery ◽  
C Elegans ◽  
Nucleolar Proteins ◽  
Multicellular Organisms

C. elegans has been an essential model organism in the fields of developmental biology, neuroscience, and aging. However, these areas have been limited by our ability to visualize and track individual C. elegans worms, especially at the subcellular scale, over the course of their lifetime. Here we present a microfluidic device to culture individual C. elegans in parallel throughout post-embryonic development. The device allows for periodic mechanical immobilization of the worm, enabling 3D imaging at subcellular precision. The immobilization is sufficient to enable fluorescence recovery after photobleaching (FRAP) measurements on organelles and other substructures within the same specific cells, throughout larval development, without the use of chemical anesthetics. Using this device, we measure FRAP recovery of two nucleolar proteins in specific intestinal cells within the same worms during larval development. We show that these exhibit different fluorescence recovery as they grow, suggesting differential protein interactions during development. We anticipate that this device will help expand the possible uses of C. elegans as a model organism, enabling its use in addressing fundamental questions at the subcellular scale, including the role of phase transitions in driving spatiotemporal intracellular organization within multicellular organisms.

Uncovering the diversity of redox proteoforms and their significance in cellular signaling and protein-protein interactions

2018 ◽  
Vol 120 ◽  
pp. S67 ◽  
Author(s):  
Eva Griesser ◽  
Uladzimir Barayeu ◽  
Jörg Flemmig ◽  
Dolores Perez-Sala ◽  
Maria Fedorova
Keyword(s):  
Protein Interactions ◽  
Cellular Signaling ◽  
Protein Protein Interactions

Extracellular cysteines C226 and C232 mediate hydrogen sulfide-dependent inhibition of Orai3-mediated store-operated calcium.

2021 ◽  
Author(s):  
Adriana M. Fresquez ◽  
Carl White
Keyword(s):  
Endoplasmic Reticulum ◽  
Hydrogen Sulfide ◽  
Protein Interactions ◽  
Fluorescence Recovery After Photobleaching ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Hek293 Cells ◽  
Protein Protein Interactions ◽  
Store Depletion ◽  
Dependent Inhibition

The gaseous signaling molecule hydrogen sulfide (H2S) physiologically regulates store-operated Ca2+ entry (SOCE). The SOCE machinery consists of the plasma membrane-localized Orai channels (Orai1-3) and endoplasmic reticulum-localized STIM1 and STIM2 proteins. H2S inhibits Orai3- but not Orai1- or Orai2-mediated SOCE. The current objective was to define the mechanism by which H2S selectively modifies Orai3. We measured SOCE and STIM1/Orai3 dynamics and interactions in HEK293 cells exogenously expressing fluorescently-tagged human STIM1 and Orai3 in the presence and absence of the H2S donor GYY4137. Two cysteines (C226 and C232) are present in Orai3 that are absent in the Orai1 and Orai2. When we mutated either of these cysteines to serine, alone or in combination, SOCE inhibition by H2S was abolished. We also established that inhibition was dependent on an interaction with STIM1. To further define the effects of H2S on STIM1/Orai3 interaction we performed a series of fluorescence recovery after photobleaching (FRAP), colocalization, and fluorescence resonance energy transfer (FRET) experiments. Treatment with H2S did not affect the mobility of Orai3 in the membrane, nor did it influence STIM1/Orai3 puncta formation or STIM1-Orai3 protein-protein interactions. These data support a model in which H2S modification of Orai3 at cysteines 226 and 232 limits SOCE evoked upon store depletion and STIM1 engagement, by a mechanism independent of the interaction between Orai3 and STIM1.

scholarly journals Differential protein-protein interactions of LRRK1 and LRRK2 indicate roles in distinct cellular signaling pathways

2014 ◽  
Vol 131 (2) ◽  
pp. 239-250 ◽  
Author(s):  
Lauran Reyniers ◽  
Maria Grazia Del Giudice ◽  
Laura Civiero ◽  
Elisa Belluzzi ◽  
Evy Lobbestael ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Protein Interactions ◽  
Cellular Signaling ◽  
Protein Protein Interactions ◽  
Differential Protein

scholarly journals The Human 2-Cys Peroxiredoxins form Widespread, Cysteine-Dependent- and Isoform-Specific Protein-Protein Interactions

Antioxidants ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 627
Author(s):  
Loes van Dam ◽  
Marc Pagès-Gallego ◽  
Paulien E. Polderman ◽  
Robert M. van Es ◽  
Boudewijn M. T. Burgering ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Cellular Signaling ◽  
Specific Protein ◽  
Large Set ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Exchange Reactions ◽  
Disulfide Exchange ◽  
Cysteine Thiols

Redox signaling is controlled by the reversible oxidation of cysteine thiols, a post-translational modification triggered by H2O2 acting as a second messenger. However, H2O2 actually reacts poorly with most cysteine thiols and it is not clear how H2O2 discriminates between cysteines to trigger appropriate signaling cascades in the presence of dedicated H2O2 scavengers like peroxiredoxins (PRDXs). It was recently suggested that peroxiredoxins act as peroxidases and facilitate H2O2-dependent oxidation of redox-regulated proteins via disulfide exchange reactions. It is unknown how the peroxiredoxin-based relay model achieves the selective substrate targeting required for adequate cellular signaling. Using a systematic mass-spectrometry-based approach to identify cysteine-dependent interactors of the five human 2-Cys peroxiredoxins, we show that all five human 2-Cys peroxiredoxins can form disulfide-dependent heterodimers with a large set of proteins. Each isoform displays a preference for a subset of disulfide-dependent binding partners, and we explore isoform-specific properties that might underlie this preference. We provide evidence that peroxiredoxin-based redox relays can proceed via two distinct molecular mechanisms. Altogether, our results support the theory that peroxiredoxins could play a role in providing not only reactivity but also selectivity in the transduction of peroxide signals to generate complex cellular signaling responses.

Protein-protein interactions of the p53 family with the Bcl-2 family in hepatocellular carcinoma

2011 ◽  
Vol 49 (08) ◽  
Author(s):  
LC König ◽  
M Meinhard ◽  
C Sandig ◽  
MH Bender ◽  
A Lovas ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
P53 Family

Partial Purification of a Specific Plasminogen Activator from Porcine Parotid Glands

1974 ◽  
Vol 31 (03) ◽  
pp. 403-414 ◽  
Author(s):  
Terence Cartwright
Keyword(s):  
Nucleic Acid ◽  
Salivary Glands ◽  
Plasminogen Activator ◽  
Protein Interactions ◽  
Partial Purification ◽  
Protein Protein Interactions ◽  
Parotid Glands ◽  
Two Stage ◽  
Preliminary Characterization ◽  
Specific Inhibitors

SummaryA method is described for the extraction with buffers of near physiological pH of a plasminogen activator from porcine salivary glands. Substantial purification of the activator was achieved although this was to some extent complicated by concomitant extraction of nucleic acid from the glands. Preliminary characterization experiments using specific inhibitors suggested that the activator functioned by a similar mechanism to that proposed for urokinase, but with some important kinetic differences in two-stage assay systems. The lack of reactivity of the pig gland enzyme in these systems might be related to the tendency to protein-protein interactions observed with this material.

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