scholarly journals Exploring cells with targeted biosensors

2016 ◽  
Vol 149 (1) ◽  
pp. 1-36 ◽  
Author(s):  
Diana Pendin ◽  
Elisa Greotti ◽  
Konstantinos Lefkimmiatis ◽  
Tullio Pozzan
Keyword(s):  
Spatial Distribution ◽  
Temporal Pattern ◽  
Cellular Signaling ◽  
Live Cells ◽  
Signaling Networks ◽  
Form Complex ◽  
Intracellular Compartments ◽  
Membrane Bound ◽  
Cellular Compartments ◽  
Protein Sensors

Cellular signaling networks are composed of multiple pathways, often interconnected, that form complex networks with great potential for cross-talk. Signal decoding depends on the nature of the message as well as its amplitude, temporal pattern, and spatial distribution. In addition, the existence of membrane-bound organelles, which are both targets and generators of messages, add further complexity to the system. The availability of sensors that can localize to specific compartments in live cells and monitor their targets with high spatial and temporal resolution is thus crucial for a better understanding of cell pathophysiology. For this reason, over the last four decades, a variety of strategies have been developed, not only to generate novel and more sensitive probes for ions, metabolites, and enzymatic activity, but also to selectively deliver these sensors to specific intracellular compartments. In this review, we summarize the principles that have been used to target organic or protein sensors to different cellular compartments and their application to cellular signaling.

scholarly journals Intracellular Ionic Strength Sensing Using NanoLuc

2021 ◽  
Vol 22 (2) ◽  
pp. 677
Author(s):  
Tausif Altamash ◽  
Wesam Ahmed ◽  
Saad Rasool ◽  
Kabir H. Biswas
Keyword(s):  
Thermal Stability ◽  
Phase Separation ◽  
Drug Discovery ◽  
Ionic Strength ◽  
Cellular Signaling ◽  
Live Cells ◽  
Protein Activity ◽  
Cellular Survival ◽  
Cellular Processes ◽  
Wide Range

Intracellular ionic strength regulates myriad cellular processes that are fundamental to cellular survival and proliferation, including protein activity, aggregation, phase separation, and cell volume. It could be altered by changes in the activity of cellular signaling pathways, such as those that impact the activity of membrane-localized ion channels or by alterations in the microenvironmental osmolarity. Therefore, there is a demand for the development of sensitive tools for real-time monitoring of intracellular ionic strength. Here, we developed a bioluminescence-based intracellular ionic strength sensing strategy using the Nano Luciferase (NanoLuc) protein that has gained tremendous utility due to its high, long-lived bioluminescence output and thermal stability. Biochemical experiments using a recombinantly purified protein showed that NanoLuc bioluminescence is dependent on the ionic strength of the reaction buffer for a wide range of ionic strength conditions. Importantly, the decrease in the NanoLuc activity observed at higher ionic strengths could be reversed by decreasing the ionic strength of the reaction, thus making it suitable for sensing intracellular ionic strength alterations. Finally, we used an mNeonGreen–NanoLuc fusion protein to successfully monitor ionic strength alterations in a ratiometric manner through independent fluorescence and bioluminescence measurements in cell lysates and live cells. We envisage that the biosensing strategy developed here for detecting alterations in intracellular ionic strength will be applicable in a wide range of experiments, including high throughput cellular signaling, ion channel functional genomics, and drug discovery.

scholarly journals Structural Basis of Complex Formation Between Mitochondrial Anion Channel VDAC1 and Hexokinase-II

2020 ◽  
Author(s):  
Nandan Haloi ◽  
Po-Chao Wen ◽  
Qunlii Cheng ◽  
Meiying Yang ◽  
Gayathri Natarajan ◽  
...  
Keyword(s):  
Complex Formation ◽  
Hybrid Approach ◽  
Md Simulations ◽  
Structural Basis ◽  
Stable Complex ◽  
Binary Complex ◽  
Hexokinase Ii ◽  
Insertion Depth ◽  
Form Complex ◽  
Membrane Bound

ABSTRACTComplex formation between hexokinase-II (HKII) and the mitochondrial channel VDAC1 plays a crucial role in regulating cell growth and survival; however, structural details of this complex remain elusive. We hypothesize that a conserved, hydrophobic helix (H-anchor) of HKII first inserts into the outer membrane of mitochondria (OMM) and then interacts with VDAC1 on the cytosolic leaflet of OMM to form a binary complex. To systematically investigate this process, we adopted a hybrid approach: 1) the membrane binding of HKII was first described with molecular dynamics (MD) simulations employing a membrane mimetic model with enhanced lipid diffusion, then 2) the resulting membrane-bound HKII was used to form complex with VDAC1 in millisecond-scale Brownian dynamics (BD) simulations. We show that H-anchor inserts its first 10 residues into the membrane, substantiating previous experimental findings. The insertion depth of the H-anchor was used to derive positional restraints in subsequent BD simulations to preserve the membrane-bound pose of HKII during the formation of the HKII/VDAC1 binary complex. Multiple BD-derived structural models were further refined with MD simulations, resulting in one stable complex. A major feature in the complex is the partial (not complete) blockade of VDAC1’s permeation pathway by HKII, a result supported by our comparative electrophysiological measurements of the channel in the presence and absence of HKII. Additionally, we showed how VDAC1 phosphorylation disrupts HKII binding, a feature that is verified by our electrophysiology recordings and have implications in mitochondria-mediated cell death.

scholarly journals Temporal sampling, resetting, and adaptation orchestrate gradient sensing in sperm

2012 ◽  
Vol 198 (6) ◽  
pp. 1075-1091 ◽  
Author(s):  
Nachiket D. Kashikar ◽  
Luis Alvarez ◽  
Reinhard Seifert ◽  
Ingo Gregor ◽  
Oliver Jäckle ◽  
...  
Keyword(s):  
Spatial Distribution ◽  
Single Molecule ◽  
Temporal Pattern ◽  
Chemical Gradient ◽  
Gradient Sensing ◽  
Temporal Sampling ◽  
Sperm Sample ◽  
Periodic Stimulation ◽  
Minimal Gradient ◽  
Sea Urchin Sperm

Sperm, navigating in a chemical gradient, are exposed to a periodic stream of chemoattractant molecules. The periodic stimulation entrains Ca2+ oscillations that control looping steering responses. It is not known how sperm sample chemoattractant molecules during periodic stimulation and adjust their sensitivity. We report that sea urchin sperm sampled molecules for 0.2–0.6 s before a Ca2+ response was produced. Additional molecules delivered during a Ca2+ response reset the cell by causing a pronounced Ca2+ drop that terminated the response; this reset was followed by a new Ca2+ rise. After stimulation, sperm adapted their sensitivity following the Weber–Fechner law. Taking into account the single-molecule sensitivity, we estimate that sperm can register a minimal gradient of 0.8 fM/µm and be attracted from as far away as 4.7 mm. Many microorganisms sense stimulus gradients along periodic paths to translate a spatial distribution of the stimulus into a temporal pattern of the cell response. Orchestration of temporal sampling, resetting, and adaptation might control gradient sensing in such organisms as well.

ATR inhibition rewires cellular signaling networks induced by replication stress

PROTEOMICS ◽  
2016 ◽  
Vol 16 (3) ◽  
pp. 402-416 ◽  
Author(s):  
Sebastian A. Wagner ◽  
Hannah Oehler ◽  
Andrea Voigt ◽  
Denis Dalic ◽  
Anja Freiwald ◽  
...  
Keyword(s):  
Cellular Signaling ◽  
Replication Stress ◽  
Signaling Networks

Quantitative multiplexed profiling of cellular signaling networks using phosphotyrosine-specific DNA-tagged SH2 domains

2006 ◽  
Vol 3 (9) ◽  
pp. 737-744 ◽  
Author(s):  
Kevin Dierck ◽  
Kazuya Machida ◽  
Anja Voigt ◽  
Julian Thimm ◽  
Martin Horstmann ◽  
...  
Keyword(s):  
Cellular Signaling ◽  
Signaling Networks ◽  
Sh2 Domains

scholarly journals Oxygen reduction in the strict anaerobe Desulfovibrio vulgaris Hildenborough: characterization of two membrane-bound oxygen reductases

Microbiology ◽  
2011 ◽  
Vol 157 (9) ◽  
pp. 2720-2732 ◽  
Author(s):  
O. Lamrabet ◽  
L. Pieulle ◽  
C. Aubert ◽  
F. Mouhamar ◽  
P. Stocker ◽  
...  
Keyword(s):  
Electron Donor ◽  
Desulfovibrio Vulgaris ◽  
Strictly Anaerobic ◽  
Quinol Oxidase ◽  
Genes Encoding ◽  
Oxygen Reductase ◽  
Membrane Bound ◽  
Carbon Monoxide Binding ◽  
Cellular Compartments ◽  
Encoding Genes

Although Desulfovibrio vulgaris Hildenborough (DvH) is a strictly anaerobic bacterium, it is able to consume oxygen in different cellular compartments, including extensive periplasmic O2 reduction with hydrogen as electron donor. The genome of DvH revealed the presence of cydAB and cox genes, encoding a quinol oxidase bd and a cytochrome c oxidase, respectively. In the membranes of DvH, we detected both quinol oxygen reductase [inhibited by heptyl-hydroxyquinoline-N-oxide (HQNO)] and cytochrome c oxidase activities. Spectral and HPLC data for the membrane fraction revealed the presence of o-, b- and d-type haems, in addition to a majority of c-type haems, but no a-type haem, in agreement with carbon monoxide-binding analysis. The cytochrome c oxidase is thus of the cc(o/b)o 3 type, a type not previously described. The monohaem cytochrome c 553 is an electron donor to the cytochrome c oxidase; its encoding gene is located upstream of the cox operon and is 50-fold more transcribed than coxI encoding the cytochrome c oxidase subunit I. Even when DvH is grown under anaerobic conditions in lactate/sulfate medium, the two terminal oxidase-encoding genes are expressed. Furthermore, the quinol oxidase bd-encoding genes are more highly expressed than the cox genes. The cox operon exhibits an atypical genomic organization, with the gene coxII located downstream of coxIV. The occurrence of these membrane-bound oxygen reductases in other strictly anaerobic Deltaproteobacteria is discussed.

scholarly journals CAPS and Munc13 utilize distinct PIP2-linked mechanisms to promote vesicle exocytosis

2014 ◽  
Vol 25 (4) ◽  
pp. 508-521 ◽  
Author(s):  
Greg Kabachinski ◽  
Masaki Yamaga ◽  
D. Michelle Kielar-Grevstad ◽  
Stephen Bruinsma ◽  
Thomas F. J. Martin
Keyword(s):  
Spatial Distribution ◽  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Total Internal Reflection ◽  
Vesicle Fusion ◽  
Live Cells ◽  
Membrane Domains ◽  
Direct View ◽  
Vesicle Exocytosis ◽  
Dependent Protein

Phosphoinositides provide compartment-specific signals for membrane trafficking. Plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) is required for Ca2+-triggered vesicle exocytosis, but whether vesicles fuse into PIP2-rich membrane domains in live cells and whether PIP2 is metabolized during Ca2+-triggered fusion were unknown. Ca2+-dependent activator protein in secretion 1 (CAPS-1; CADPS/UNC31) and ubMunc13-2 (UNC13B) are PIP2-binding proteins required for Ca2+-triggered vesicle exocytosis in neuroendocrine PC12 cells. These proteins are likely effectors for PIP2, but their localization during exocytosis had not been determined. Using total internal reflection fluorescence microscopy in live cells, we identify PIP2-rich membrane domains at sites of vesicle fusion. CAPS is found to reside on vesicles but depends on plasma membrane PIP2 for its activity. Munc13 is cytoplasmic, but Ca2+-dependent translocation to PIP2-rich plasma membrane domains is required for its activity. The results reveal that vesicle fusion into PIP2-rich membrane domains is facilitated by sequential PIP2-dependent activation of CAPS and PIP2-dependent recruitment of Munc13. PIP2 hydrolysis only occurs under strong Ca2+ influx conditions sufficient to activate phospholipase Cη2 (PLCη2). Such conditions reduce CAPS activity and enhance Munc13 activity, establishing PLCη2 as a Ca2+-dependent modulator of exocytosis. These studies provide a direct view of the spatial distribution of PIP2 linked to vesicle exocytosis via regulation of lipid-dependent protein effectors CAPS and Munc13.

Intracellular distribution of cations and protein in regenerating rat liver

1963 ◽  
Vol 204 (2) ◽  
pp. 257-261 ◽  
Author(s):  
Edwin M. Uyeki
Keyword(s):  
Liver Regeneration ◽  
Temporal Pattern ◽  
Initial Phase ◽  
Intracellular Distribution ◽  
Potassium Ions ◽  
Sodium Ions ◽  
Protein Distribution ◽  
Intracellular Compartments ◽  
Two Phases ◽  
Early Phases

The intracellular distribution of several cell constituents was studied at different periods after partial hepatectomy in the rat. The temporal pattern of cation and protein distribution can be divided into two phases: there is an initial phase in the first 24 hr posthepatectomy in which sodium and water increased in tissues concomitant with a decrease in potassium and protein. The levels of these several constituents subsequently approached values that were higher than in the control at 1 and 2 days posthepatectomy and were generally maintained at these values throughout the assay period. The reasons for the accumulation of higher than normal levels of both potassium and sodium are as yet uninterpreted; the higher than control potassium-to-sodium ratios generally observed in the intracellular compartments are due to the relative greater accumulation of potassium ions over sodium ions during this period. Comparing the intracellular compartments, the greatest alterations of constituents in the early phases of liver regeneration were observed in the soluble portion.

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