Cell Cycle Dependent Modulation of Membrane Dipole Potential and Neurotransmitter Receptor Activity: Role of Membrane Cholesterol

2020 ◽  
Vol 11 (18) ◽  
pp. 2890-2899
Author(s):  
Parijat Sarkar ◽  
Bhagyashree D. Rao ◽  
Amitabha Chattopadhyay
Keyword(s):  
Cell Cycle ◽  
Receptor Activity ◽  
Membrane Cholesterol ◽  
Dipole Potential ◽  
Membrane Dipole Potential ◽  
Neurotransmitter Receptor ◽  
Cycle Dependent

A direct measurement of increased divalent cation influx in fertilised mouse oocytes

Development ◽  
1996 ◽  
Vol 122 (7) ◽  
pp. 2199-2206 ◽  
Author(s):  
O.M. McGuinness ◽  
R.B. Moreton ◽  
M.H. Johnson ◽  
M.J. Berridge
Keyword(s):  
Cell Cycle ◽  
Dependent Manner ◽  
Continuous Increase ◽  
Mouse Oocytes ◽  
A Cell ◽  
Cycle Dependent ◽  
Precise Role ◽  
Entry Mechanism ◽  
Stimulation Of

On fertilisation of mouse oocytes, the fusing spermatozoon triggers a series of repetitive calcium (Ca2+) spikes. The Ca2+ spikes seem to be necessary for successful progression through the cell cycle and are regulated in a cell-cycle-dependent manner. The spikes appear to require the linkage of continuous Ca2+ influx to the periodic release of Ca2+ from intracellular stores by a process of Ca(2+)-induced Ca2+ release. The precise role of Ca2+ influx was explored using the manganese (Mn2+)-quench technique to monitor unidirectional cation influx into single mouse oocytes. There was a marked stimulation of cation influx associated closely with the upsweep of the first and subsequent fertilisation Ca2+ spikes. A smaller but significant increase in the rate of cation influx persisted in the interspike period in fertilised oocytes. Spike-associated entry was not as apparent in oocytes stimulated to spike repetitively by thimerosal or acetylcholine application. Instead, there was a continuous increase in cation influx underlying Ca2+ spiking which commenced with the onset of the first spike. Using the specific microsomal inhibitor thapsigargin and the Ca2+ ionophore ionomycin, we found evidence for a capacitative entry mechanism in mouse oocytes. We propose that the persistent influx of Ca2+ observed in response to all stimuli examined is controlled by a capacitative mechanism and sets the frequency of spiking by determining the time taken to refill the internal stores to a point where they are again sensitive enough to initiate the next spike.

A Role for NIPA in DNA Damage Response.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1265-1265
Author(s):  
Christine von Klitzing ◽  
Florian Bassermann ◽  
Stephan W. Morris ◽  
Christian Peschel ◽  
Justus Duyster
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Phosphorylation Site ◽  
Genotoxic Stress ◽  
S Phase ◽  
Damage Response ◽  
A Cell ◽  
Cycle Dependent

Abstract The nuclear interaction partner of ALK (NIPA) is a nuclear protein identified by our group in a screen for NPM-ALK interaction partners. We recently reported that NIPA is an F-box protein that assembles with SKP1, Cul1 and Roc1 to establish a novel SCF-type E3 ubiquitin ligase. The formation of the SCFNIPA complex is regulated by cell cycle-dependent phosphorylation of NIPA that restricts SCFNIPA assembly from G1- to late S-phase, thus allowing its substrates to be active from late S-phase throughout mitosis. Proteins involved in cell cycle regulation frequently play a role in DNA damage checkpoints. We therefore sought to determine whether NIPA has a function in the cellular response to genotoxic stress. For this reason we treated NIH/3T3 cells with various DNA-damaging agents. Surprisingly, we observed phosphorylation of NIPA in response to some of these agents, including UV radiation. This phosphorylation was cell cycle phase independent and thus independent of the physiological cell cycle dependent phosphorylation of NIPA. The relevant phosphorylation site is identical to the respective site in the course of cell cycle-dependent phosphorylation of NIPA. Thus, phosphorylation of NIPA upon genotoxic stress would inactivate the SCFNIPA complex in a cell cycle independent manner. Interestingly, this phosphorylation site lies within a consensus site of the Chk1/Chk2 checkpoint kinases. These kinases are central to DNA damage checkpoint signaling. Chk1 is activated by ATR in response to blocked replication forks as they occur after treatment with UV. We performed experiments using the ATM/ATR inhibitor caffeine and the Chk1 inhibitor SB218078 to investigate a potential role of Chk1 in NIPA phosphorylation. Indeed, we found both inhibitors to prevent UV-induced phosphorylation of NIPA. Current experiments applying Chk1 knock-out cells will unravel the role of Chk1 in NIPA phosphorylation. Additional experiments were performed to investigate a function for NIPA in DNA-damage induced apoptosis. In this regard, we observed overexpression of NIPA WT to induce apoptosis in response to UV, whereas no proapoptotic effect was seen with the phosphorylation deficient NIPA mutant. Therefore, the phosphorylated form of NIPA may be involved in apoptotic signaling pathways. In summary, we present data suggesting a cell cycle independent function for NIPA. This activity is involved in DNA damage response and may be involved in regulating apoptosis upon genotoxic stress.

scholarly journals Cell cycle-dependent phosphorylation and dephosphorylation of the yeast DNA polymerase alpha-primase B subunit.

1995 ◽  
Vol 15 (2) ◽  
pp. 883-891 ◽  
Author(s):  
M Foiani ◽  
G Liberi ◽  
G Lucchini ◽  
P Plevani
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Posttranslational Modifications ◽  
S Phase ◽  
Dna Polymerase Alpha ◽  
B Subunit ◽  
Initiation Of Dna Replication ◽  
Cycle Dependent

The yeast DNA polymerase alpha-primase B subunit functions in initiation of DNA replication. This protein is present in two forms, of 86 and 91 kDa, and the p91 polypeptide results from cell cycle-regulated phosphorylation of p86. The B subunit present in G1 arises by dephosphorylation of p91 while cells are exiting from mitosis, becomes phosphorylated in early S phase, and is competent and sufficient to initiate DNA replication. The B subunit transiently synthesized as a consequence of periodic transcription of the POL12 gene is phosphorylated no earlier than G2. Phosphorylation of the B subunit does not require execution of the CDC7-dependent step and ongoing DNA synthesis. We suggest that posttranslational modifications of the B subunit might modulate the role of DNA polymerase alpha-primase in DNA replication.

scholarly journals Cdk1 phosphorylation of the dynein adapter Nde1 controls cargo binding from G2 to anaphase

2018 ◽  
Vol 217 (9) ◽  
pp. 3019-3029 ◽  
Author(s):  
Caitlin L. Wynne ◽  
Richard B. Vallee
Keyword(s):  
Cell Cycle ◽  
Phosphorylation Site ◽  
Cytoplasmic Dynein ◽  
In Vitro Assays ◽  
Cargo Protein ◽  
Specific Association ◽  
Cycle Dependent ◽  
Cargo Binding

Cytoplasmic dynein is involved in diverse cell cycle–dependent functions regulated by several accessory factors, including Nde1 and Ndel1. Little is known about the role of these proteins in dynein cargo binding, and less is known about their cell cycle­–dependent dynein regulation. Using Nde1 RNAi, mutant cDNAs, and a phosphorylation site–specific antibody, we found a specific association of phospho-Nde1 with the late G2-M nuclear envelope and prophase to anaphase kinetochores, comparable to the pattern for the Nde1 interactor CENP-F. Phosphomutant-Nde1 associated only with prometaphase kinetochores and showed weaker CENP-F binding in in vitro assays. Nde1 RNAi caused severe delays in mitotic progression, which were substantially rescued by both phosphomimetic and phosphomutant Nde1. Expression of a dynein-binding–deficient Nde1 mutant reduced kinetochore dynein by half, indicating a major role for Nde1 in kinetochore dynein recruitment. These results establish CENP-F as the first well-characterized Nde1 cargo protein, and reveal phosphorylation control of Nde1 cargo binding throughout a substantial fraction of the cell cycle.

scholarly journals C-Glucosylation as a tool for the prevention of PAINS-induced membrane dipole potential alterations

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ana Marta de Matos ◽  
Maria Teresa Blázquez-Sánchez ◽  
Carla Sousa ◽  
Maria Conceição Oliveira ◽  
Rodrigo F. M. de Almeida ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Transmembrane Protein ◽  
New Technology ◽  
Dipole Potential ◽  
Membrane Dipole Potential ◽  
Cellular Processes ◽  
Established Relationship ◽  
And Function ◽  
Bioactive Natural Product

AbstractThe concept of Pan-Assay Interference Compounds (PAINS) is regarded as a threat to the recognition of the broad bioactivity of natural products. Based on the established relationship between altered membrane dipole potential and transmembrane protein conformation and function, we investigate here polyphenols' ability to induce changes in cell membrane dipole potential. Ultimately, we are interested in finding a tool to prevent polyphenol PAINS-type behavior and produce compounds less prone to untargeted and promiscuous interactions with the cell membrane. Di-8-ANEPPS fluorescence ratiometric measurements suggest that planar lipophilic polyphenols—phloretin, genistein and resveratrol—act by decreasing membrane dipole potential, especially in cholesterol-rich domains such as lipid rafts, which play a role in important cellular processes. These results provide a mechanism for their labelling as PAINS through their ability to disrupt cell membrane homeostasis. Aiming to explore the role of C-glucosylation in PAINS membrane-interfering behavior, we disclose herein the first synthesis of 4-glucosylresveratrol, starting from 5-hydroxymethylbenzene-1,3-diol, via C-glucosylation, oxidation and Horner-Wadsworth-Emmons olefination, and resynthesize phloretin and genistein C-glucosides. We show that C-glucosylation generates compounds which are no longer able to modify membrane dipole potential. Therefore, it can be devised as a strategy to generate bioactive natural product derivatives that no longer act as membrane dipole potential modifiers. Our results offer a new technology towards rescuing bioactive polyphenols from their PAINS danger label through C–C ligation of sugars.

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